The confidence level of Hong Kong’s exporters has fallen to its lowest-ever level in the face of a triple challenge – the COVID-19 outbreak, softening global demand and lingering trade tension between the United States and Mainland...

Guest of Honour at Film Mayo launch on 1st December

The Lands Department will provide additional rental or fee concessions for tenants of short-term tenancies (STT) and waiver holders in accordance with the new round of support measures announced earlier this month.

The measures aim to strengthen support for enterprises in light of the challenges arising from the COVID-19 epidemic.

In accordance with the support measures launched in 2019 and those announced in the 2020-21 Budget, STTs and waivers for varying the terms of land grants for business and community uses under the department have been granted 50% rental or fee concession from October 2019 to September this year.

Under the new round of support measures, the 5,000 eligible STT tenants and waiver holders already enjoying the previous concession, such as catering facilities, shops, workshops, public fee-paying car parks, and welfare facilities, will see their rental or fee concession rate increase to 75% from April to September.

The 75% concession arrangement will be extended to businesses not covered previously, such as depots for public transport operators, public utilities, petrol filling stations, driving schools and advertising facilities, effective for the same period.

If these tenants and waiver holders are ordered to close or have chosen to close due to the Government’s orders or other restrictions for safeguarding public health under the relevant regulation, they may further apply to the department for full rental or fee concession for the duration of the closure.

The department will issue written notifications to eligible STT tenants and waiver holders on the above concession measures and their implementation details.           

Additionally, to help development projects with construction progress affected by the epidemic, the department will offer extensions of the Building Covenant period at nil premium for up to six months for leases with the covenant not yet discharged as at April 8.

The department will issue a practice note and upload it onto its website by the end of the month to announce the application arrangements for lessees.

Gretchen Benson
Feb 1, 2011; 24:36-40
Nutrition FYI

Ekta Singh
Nov 1, 2012; 25:217-221
From Research to Practice

Mays H. Vue
Aug 1, 2011; 24:171-177
Pharmacy and Therapeutics

Amy P. Campbell
May 1, 2017; 30:76-81
From Research to Practice

Abdur Rehman
Nov 1, 2011; 24:234-238
Pharmacy and Therapeutics

Peggy Yarborough
Jan 1, 2003; 16:
Case Studies

Curtis Triplitt
Oct 1, 2006; 19:202-211
Pharmacy Update

The Food & Environmental Hygiene Department announced that applications for the Catering Business (Social Distancing) Subsidy Scheme, under the second round of the Anti-epidemic Fund, will start from May 5.

The department said the scheme, which is estimated to benefit 16,000 catering outlets and their employees, will provide financial relief measures to the catering businesses which have been hard hit by the COVID-19 epidemic and social distancing measures.

It will provide subsidies ranging from $250,000 to $2,200,000 to eligible licence holders of general restaurants, light refreshment restaurants, marine restaurants and factory canteens in operation according to the floor area of the premises as specified on the licence.

The primary goal of the scheme is to support the payment of employees' salaries during a six-month period following application approval.

To provide immediate relief to the catering sector, the subsidy will be dished out upfront, in two tranches.

To avoid abuse, applicants should undertake that there will be no redundancy of staff for three months on receipt of the first tranche of subsidy and another three months on receipt of the second tranche.

They should also undertake that not less than 80% of the subsidies for respective months would be used to pay salaries of staff working at the premises.

Applicants should submit within a specified period a certificate issued by a Certified Public Accountant (practising) on its total staff salaries and total number of salaried staff for each of the months covered by the subsidy.

They should also submit a certificate issued by a CPA on its total staff salaries and total number of staff working at the premises for the month of March this year.

To avoid double benefits, applicants should declare that they have not and will not submit any application under the Employment Support Scheme.

Each catering outlet directed to close its licensed premises under the Prevention & Control of Disease (Requirements & Directions) (Business & Premises) Regulation, including karaoke establishments, nightclubs and bars or pubs, is eligible for a further one-off subsidy of $50,000.

Applications should be submitted to the department's District Environmental Hygiene Offices in person or by mail. Food licence holders can also submit their applications through the website.

The deadline for application is June 5.

Upon receipt of the completed application form and supporting documents and after verification, the disbursement of the first tranche of subsidies can generally be made in two to three weeks by crossed cheques to the corresponding licensed food premises.

There have been arrests, prosecutions and convictions of youngsters and students resulting from the social unrest since June last year. Recently, explosives and chemicals were found by Police. It would be catastrophic if they were used for unscrupulous purposes. Some of the youngsters might have been mistakenly led to believe that use of violence should be considered as a means to achieve their aspirations. However, I hope that they appreciate that law-breaking behaviour would always have legal consequences.

It is very troubling to see from the media reports that underage children or students are taking part in unlawful activities and even getting involved in violence. In so doing, they would not only put their own and others' safety at risk, but could also be arrested and prosecuted resulting in criminal records upon convictions. Their future in education and employment would inevitably be affected.

There are misleading suggestions that all young offenders would only be cautioned under the Police Superintendent's Discretion Scheme without criminal records. One must understand that Police, in deliberating whether to administer a caution, have to take into account a number of factors, such as whether the juvenile offender was under the age of 18 at the time of committing the offence; the seriousness and nature of the offence; whether the offender voluntarily and unequivocally admits the offence; whether the offender is remorseful and so on. Therefore, the scheme does not apply to all young offenders indiscriminately.

Similarly, bind over orders are only appropriate for first-time offenders involving minor offences and they have to be agreed by the courts. Under the bind over arrangement, offenders are required to admit the wrongdoings and are admonished in an open court. They also need to give an undertaking to the court to be of good behaviour. Imprisonment might be imposed for breaching the undertaking. Before agreeing to such a procedure on specified conditions, the prosecution has to give considerations to various factors including the age of the offender, criminal record, and attitude of the offender such as remorsefulness and so on.

There are also comments that prosecutions against young offenders should be withdrawn on the basis of public interest. I have to stress again that the Department of Justice (DoJ) must strictly make our prosecutorial decisions in accordance with admissible evidence, applicable laws and the Prosecution Code. The non-exhaustive list of considerations to be addressed when making assessment in considering public interest, including those applicable to juvenile offenders, were set out in 5.9 and 15.4 of the Prosecution Code, which include the nature of the offence, the seriousness of the offence, the criminal history of the suspect, remorsefulness of the suspect and the sentencing options available to the court and so on. Prosecutors should follow the above principles in making their decisions and must not withdraw charges unjustifiably under the pretext of public interest.

In considering the above arrangements, the DoJ has to carefully examine the circumstances of each case in an objective manner and strictly in accordance with the established principles. A decision cannot be made solely based on the age of the offenders. Their legal representatives are not only duty bound to inform them of their rights but also have an obligation to remind them of the legal consequences and the availability and considerations in relation to the above arrangements.

As observed by Lord Diplock, "[t]he acceptance of the rule of law as a constitutional principle requires that a citizen, before committing himself to any course of action, should be able to know in advance what are the legal consequences that will flow from it". Every member of the public must be aware that one has to bear the legal responsibility resulting from law-breaking behaviour. I sincerely hope that every one, in particular the youngsters, should exercise their independent thinking and judgement, and, more importantly, stay away from violence and not commit mistakes that might ruin their lives in the future.

Secretary for Justice Teresa Cheng wrote this article and posted it on her blog on May 9.

(Rice University) Rice University researchers have integrated high-efficiency solar cells and electrode catalysts into an efficient, low-cost device that splits water to produce hydrogen fuel.

(Ecole Polytechnique Fédérale de Lausanne) EPFL chemists have developed sponges to capture various target substances, like gold, mercury and lead, dissolved in solution. The sponges are actually porous crystals called metal organic frameworks, and now one exists for capturing toxic hexavalent chromium from water.

(University of Jyväskylä - Jyväskylän yliopisto) Researchers at the universities of Jyvaskyla and Xiamen discovered a novel way to make functional macroscopic crystalline materials out of nanometer-size 34-atom silver-gold intermetallic clusters. The cluster material has a highly anisotropic electrical conductivity, being a semiconductor in one direction and an electrical insulator in other directions. The research was published in Nature Communications on May 6, 2020.

(Johannes Gutenberg Universitaet Mainz) Scientists at Johannes Gutenberg University Mainz (JGU) and the Helmholtz Institute Mainz (HIM) in Germany have presented a non-contact method for detecting the state of charge and any defects in lithium-ion batteries.

(Lancaster University) Lancaster University's Dr Samuli Autti has been awarded a Young Scientist Prize 2020 by the International Union of Pure and Applied Physics. The prestigious prize, awarded only once every three years, was made by the Low Temperature Commission of the IUPAP.

The Innovation & Technology Bureau announced that as of 3pm today, the CuMask online registration system had received over 500,000 registrations, covering close to 1.38 million registrants in total. 

The bureau also responded to reports concerning the purpose of information collection and security of the registration system.

It noted that information provided by citizens in obtaining the masks will not be used for other purposes and that the Government will ensure the retention period of the personal data is no longer than the time required for the purposes for which the data is used.

The bureau pointed out that the registration system for masks operates on the Government's private cloud to ensure the stability and security of the system. 

In order to prevent intrusion and data leakage, multiple security measures have been put in place in compliance with the Government Information Technology Security Policy & Guidelines. 

These measures include a firewall, intrusion detection, anti-bot technology and installation of the latest anti-virus software with regular updates of virus definitions. 

The service has also passed an information security risk assessment and audit before launch.

The Privacy Commissioner for Personal Data has been consulted on the system's personal data processing arrangements. 

The system has also passed an independent third-party privacy impact assessment to ensure that the relevant service and system comply with the Personal Data (Privacy) Ordinance.

The bureau further explained that citizens need to provide their Hong Kong identity card number and date of birth for the registration system to match data with the Immigration Department’s system.

The process will be used to ascertain whether the registrant is a Hong Kong resident and check against any duplicated registrations. 

The local mobile number serves as a way to receive SMS messages on registration results and delivery, while the name and local address of the main registrant serves to verify whether the address exists and for arranging delivery. 

The bureau emphasised that the purposes of information collection have been clearly displayed on the front of the registration page for citizens to browse before registration.

Meanwhile, the bureau clarified the online rumours regarding the manufacturer of CuMask, noting that the CuMask is not manufactured by the Sun Hing Knitting Factory Limited nor Nan Fung Group.

The procurement of raw materials, coordination of production, sterilisation and packaging of the CuMask are being handled by the Hong Kong Research Institute of Textiles & Apparel, it said.

The bureau expressed regret about the rumours.

The CuMask online registration system received over 720,000 registrations, covering two million registrants in total on the first day of registration on May 6, the Innovation & Technology Bureau announced today.

The bureau said the response is overwhelming and it is encouraged to see support for local invention.

"Our thanks go to support from all sides, including the Hong Kong Research Institute of Textiles & Apparel (HKRITA) which has been commissioned to oversee the project, the Crystal International Group Limited which is responsible for the production, the Novetex Textiles Limited in Tai Po Industrial Estate for providing clean room for sterilisation, The Mills and the TAL Apparel Limited for lending premises to set up workshops as well as the frontline workmen for their hard work over the past few months.

“This unrivalled challenge cannot be met without their joint efforts and the collaboration of the industry and our team,” the bureau stated.

The bureau commissioned the HKRITA to oversee the CuMask project in order to meet the imminent needs for masks in Hong Kong.

It pointed out that the Government Stores & Procurement Regulations do allow direct purchase to be made under extreme urgency.

The whole procurement process was conducted in accordance with the Government's procurement regulations and procedures and with confirmation that the conditions under the Agreement on Government Procurement of the World Trade Organization could be met.

The bureau further explained that in February and March this year, the Government contacted various suppliers of reusable masks. However, most stated that they had either stopped production, did not have enough stock, were unable to export materials due to export control, or unable to produce testing certification.

The epidemic at that time was serious and the supplies of anti-epidemic items were becoming scarce. Hong Kong did not have any raw materials or production lines.

Taking into account the aggressive procurement actions of anti-epidemic items by different countries, export control and suspension of production lines all over the world, the Government had to consider urgently the feasibility of manufacturing reusable masks that would be up to standard for use by the whole community.

On reviewing the reusable mask developed by the HKRITA, the Government considered that the design of the mask and materials used could meet the requirement, as there were supporting certifications proving its compliance with relevant international standards.

As for mass production, it depends on the availability of supply of raw materials. Having wide network in the industry, the HKRITA was able to acquire quality raw materials within a short period and put production lines in place.

The Government therefore commissioned the HKRITA to oversee the coordination of production through direct purchase with a view to supplying reusable masks to all Hong Kong residents as soon as possible.

The bureau added the HKRITA is a non-profit-making R&D centre fully subsidised by the Government, with most of their R&D projects funded by the Innovation & Technology Fund.

The HKRITA oversees the CuMask project on a non-profit-making basis. All expenses will be reimbursed to the HKRITA on the basis of actual spending.

People concerned with the effectiveness of the CuMask may browse the website for testing reports and patent information.

(To watch the full press briefing with sign language interpretation, click here.)

The Centre for Health Protection today said recovered COVID-19 patients or those who did not have any symptoms may be discharged from hospital 10 days after the onset of symptoms or a positive test result.

Its Communicable Disease Branch Head Dr Chuang Shuk-kwan told a press briefing that the revised discharge criteria was based on the latest scientific evidence.

“Our Scientific Committee on Emerging & Zoonotic Diseases met yesterday and examined the latest scientific evidence on whether the virus will be viable from a patient.

“And the available evidence showed that this virus is usually not detected after 10 days since the onset of symptoms of patients. Some patients may have persistent positive PCR (polymerase chain reaction) for a long period of time.”

Dr Chuang noted that patients still had to meet the criteria of having two clinical specimens test negative, or testing positive for the SARS-CoV-2 antibody to be discharged.

“We have revised the discharge criteria to include the patient who (must have) been staying in the hospital for at least 10 days after the onset of symptoms. So this is the additional criteria, in addition to the previous criteria of two consecutive negative specimens.

“We added another criteria (which is) in case a patient has stayed in the hospital for a long time, more than 10 days since the onset of symptoms, but he or she has persistent positive PCR despite the Ct (cycle threshold) value being very high, they can check their serology, the antibody. So if the antibody turns positive, usually it is after 10 days, then he or she can be discharged.

“So this is based on the latest scientific evidence.”

"In his new book--The Math of Life & Death: 7 Mathematical Principles that Shape Our Lives--mathematician Kit Yates makes complex mathematical concepts easily accessible to anyone, and which can improve decision making in an increasingly quantitative society. In this Q&A, Yates discusses why math is relevant to everyday life." See "Mathematician Kit Yates on Anti Vaxxer Movement, Air Travel Germs and Samoa's Measles Outbreak," by Meredith Wold Schizer, Newsweek, December 23, 2019.

Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signaling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP down-regulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-HPLC-tandem MS (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells, and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also down-regulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signaling protein extracellular signal-regulated kinase, and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP down-regulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity.

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro. β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography–tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.

Sulfur is essential for biological processes such as amino acid biogenesis, iron–sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer–substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine–bound FliY, and maximally by l-cysteine– or l-cystine–bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein–coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family–specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.

Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and short hairpin RNA–based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel–dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea32+ and CuB1+, and suggests that a peroxide-bound state (Fea33+–O−–O−–CuB2+) rather than an O2-bound state (Fea32+–O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fea32+–O2, whereas Fea33+–O−–O−–CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fea34+ = O2−, not as Fea34+–OH−. The distance suggests an ∼800-cm−1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+–O−–O−–CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β-fold, followed by a secretin/TonB, short N-terminal subdomain at the C terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pulldown assays, isothermal titration calorimetry, and thermal denaturation CD spectroscopy, we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation.

The Mycobacterium tuberculosis virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear. In the present study, using liposome-leakage and cytotoxicity assays, LC-MS/MS–based proteomics, and CCF-4 FRET analysis, we obtained evidence that the Nα-acetylation of the Thr-2 residue on EsxA, a post-translational modification that is present in mycobacteria but absent in Escherichia coli, is required for the EsxA:B separation. Substitutions at Thr-2 that precluded Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamics simulations revealed that at low pH, the Nα-acetylated Thr-2 makes direct and frequent “bind-and-release” contacts with EsxB, which generates a force that pulls EsxB away from EsxA. In summary, our findings provide evidence that the Nα-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine–ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure–function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase–ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein–cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin–γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens–epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone–client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone–client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin–client complexes could contribute to lens aging and presbyopia.

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5–ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5–ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5–ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

Fabry disease is a heritable lipid disorder caused by the low activity of α-galactosidase A and characterized by the systemic accumulation of globotriaosylceramide (Gb3). Recent studies have reported a structural heterogeneity of Gb3 in Fabry disease, including Gb3 isoforms with different fatty acids and Gb3 analogs with modifications on the sphingosine moiety. However, Gb3 assays are often performed only on the selected Gb3 isoforms. To precisely determine the total Gb3 concentration, here we established two methods for determining both Gb3 isoforms and analogs. One was the deacylation method, involving Gb3 treatment with sphingolipid ceramide N-deacylase, followed by an assay of the deacylated products, globotriaosylsphingosine (lyso-Gb3) and its analogs, by ultra-performance LC coupled to tandem MS (UPLC-MS/MS). The other method was a direct assay established in the present study for 37 Gb3 isoforms and analogs/isoforms by UPLC-MS/MS. Gb3s from the organs of symptomatic animals of a Fabry disease mouse model were mainly Gb3 isoforms and two Gb3 analogs, such as Gb3(+18) containing the lyso-Gb3(+18) moiety and Gb3(−2) containing the lyso-Gb3(−2) moiety. The total concentrations and Gb3 analog distributions determined by the two methods were comparable. Gb3(+18) levels were high in the kidneys (24% of total Gb3) and the liver (13%), and we observed Gb3(−2) in the heart (10%) and the kidneys (5%). These results indicate organ-specific expression of Gb3 analogs, insights that may lead to a deeper understanding of the pathophysiology of Fabry disease.

VOLUME 295 (2020) PAGES 1898–1914Yichen Zhong's name was misspelled. The correct spelling is shown above.

Although a robust inflammatory response is needed to combat infection, this response must ultimately be terminated to prevent chronic inflammation. One mechanism that terminates inflammatory signaling is the production of alternative mRNA splice forms in the Toll-like receptor (TLR) signaling pathway. Whereas most genes in the TLR pathway encode positive mediators of inflammatory signaling, several, including that encoding the MyD88 signaling adaptor, also produce alternative spliced mRNA isoforms that encode dominant-negative inhibitors of the response. Production of these negatively acting alternatively spliced isoforms is induced by stimulation with the TLR4 agonist lipopolysaccharide (LPS); thus, this alternative pre-mRNA splicing represents a negative feedback loop that terminates TLR signaling and prevents chronic inflammation. In the current study, we investigated the mechanisms regulating the LPS-induced alternative pre-mRNA splicing of the MyD88 transcript in murine macrophages. We found that 1) the induction of the alternatively spliced MyD88 form is due to alternative pre-mRNA splicing and not caused by another RNA regulatory mechanism, 2) MyD88 splicing is regulated by both the MyD88- and TRIF-dependent arms of the TLR signaling pathway, 3) MyD88 splicing is regulated by the NF-κB transcription factor, and 4) NF-κB likely regulates MyD88 alternative pre-mRNA splicing per se rather than regulating splicing indirectly by altering MyD88 transcription. We conclude that alternative splicing of MyD88 may provide a sensitive mechanism that ensures robust termination of inflammation for tissue repair and restoration of normal tissue homeostasis once an infection is controlled.

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain–mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain–mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain–mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain–mediated complex formation and provide mechanistic insights into how SARAH domain–mediated interactions influence Hippo pathway activity.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA–binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5'- and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. Mycobacterial type VII secretion systems consist of five subtypes, ESX-1–5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum. Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS–based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9th carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo. Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.

(American Association for the Advancement of Science) A one-hour exercise designed to increase feelings of social belonging administered during the first year of college appears to significantly improve the lives and careers of black students up to 11 years later, psychologists report.