(College of Engineering, Carnegie Mellon University) Carnegie Mellon University's Maarten de Boer and Gianluca Piazza are developing reliable, mechanical switches the size of a DNA molecule, thanks to a $2M LEAP-HI grant from the National Science Foundation.

(University of Illinois Grainger College of Engineering) Three Nick Holonyak Jr., Micro and Nanotechnology Lab (HMNTL) faculty members received NSF Rapid Response Research (RAPID) program grants, all of which aim to shorten the amount of time it takes to process a COVID-19 test with less false negatives. Current tests can take as long as five days for results to be.

(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.

Claus Sorensen
Represent. Theory 24 (2020), 151-177.
Abstract, references and article information

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

Chief Executive Carrie Lam today said that Hong Kong’s needy and disadvantaged will receive masks as part of the Government’s new mask distribution programme.

Mrs Lam made the announcement during a press conference this afternoon and explained that millions of donated masks will also be distributed to those in need.

“I have outlined six measures to distribute masks freely to the people of Hong Kong, and of course in so doing, we will take special account of the disadvantaged, the elderly and street sleepers. 

“So apart from being a member of the Hong Kong population, they will receive reusable masks. They will receive disposable masks. We have this mask distribution program together with a large number of non-governmental organisations, charity groups and self-help groups.

“So we will continue to work with them to distribute another three million masks, which were donated to us.”

Mrs Lam emphasised that should there be a shortage of masks set aside for the needy, the Government will use its own supply to cover the shortfall.

“I am announcing that if we run out of donated masks, but there is still a need from this disadvantaged groups, we will use the government masks - the masks that we procured which are supposed to be for our own use - and share these with the needy groups in society.

“That's a way to ensure that, in a public health situation that we are now in, the needs of the disadvantaged groups will be fully taken care of.”

In addition to distributing donated masks, the Government announced other measures on mask supplies that include handing out single-use and reusable masks to all Hong Kong residents and students in need.

Such measures also call for increasing the supply of masks to staff of elderly homes and cleaning workers employed by the Government's outsourced service contractors, as well as providing masks to private medical practitioners.

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 Government urged District Councillors to focus on livelihood issues and discuss matters rationally, adding that it will continue to co-operate with the District Council under the principles of mutual respect, observation of order and rational discussion.

The Government issued the statement after a number of Central & Western District Council members today entered the office area of the Central & Western District Office without consent.

The statement noted that the members shouted loudly and knocked on the door of the office.

Despite repeated responses and an appeal from the District Office staff, the members still refused to leave.

The statement added that the members stayed in the District Office for a long time, seriously affecting its operation.

The Government expressed regret over their acts.

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.

More than 8,200 applications for the Anti-epidemic Support Scheme for Property Management Sector (ASPM) have been received, with over 3,850 approved, the Government announced today.

The approved applications involve subsidies of more than $140 million and will benefit around 22,000 building blocks and about 35,750 frontline property management workers.

Launched under the Anti-epidemic Fund, the ASPM provides subsidies to owners' organisations or property management companies of eligible buildings to provide hardship allowance to frontline property management workers.

It also provides the Anti-epidemic Cleansing Subsidy to owners' organisations or property management companies.

The scheme’s first phase covers private residential and composite buildings, while its second phase covers industrial and commercial buildings.

The ASPM is still open for applications and continues to disburse subsidies.

Contact the Property Management Services Authority at 3696 1156 or 3696 1166, or visit its website for details.

Students in a math class at Columbine High School in Colorado used geometry to work with Habitat for Humanity to build homes for those in need. See the video segment at "Students Build Houses For Families In Need...In Math Class," by Shaun Boyd, CBS4 Denver TV, December 23, 2019.

IntroductionThe nucleolus is the central organelle within eukaryotic cells whose primary function is to generate ribosomes, the major protein producing machines within all cells. New roles for the nucleolus are continuously emerging as we explore its molecular intricacies. Despite the central and fundamental role of the nucleolus in cell biology, there has previously been no single official meeting that enables the gathering of scientists whose research converges on the nucleolus. As a result, the community of researchers who study this organelle risks fragmentation across disciplines. The Emerging Roles for the Nucleolus Symposium, which has now taken place twice on a biennial basis, first in 2017 (1) and again in 2019, therefore, represents the first of its kind. The overarching goals of this symposium are (a) to convene researchers who study the nucleolus across model systems (yeast, nematodes, fruit flies, mouse, human cell lines) and biological perspectives (structural, biophysical, molecular, cellular, pathophysiology), (b) to share and disseminate the latest research breakthroughs in nucleolar biology, (c) to promote interaction, engagement, and collaboration centered on the nucleolus across disciplines, and (d) to provide trainees and early career investigators with an organelle-specific scientific community of support.The second Emerging Roles for the Nucleolus meeting was sponsored by the American Society for Biochemistry and Molecular Biology and was held at the Stowers Institute for Medical Research in Kansas City, MO, from October 24 to October 27, 2019. It was organized by Jennifer Gerton (Stowers Institute), Francesca Duncan (Northwestern University Feinberg School of Medicine), and Craig Pikaard...

Telomeres are specific nucleoprotein structures that are located at the ends of linear eukaryotic chromosomes and play crucial roles in genomic stability. Telomere DNA consists of simple repeats of a short G-rich sequence: TTAGGG in mammals and TTTAGGG in most plants. In recent years, the mammalian telomeric G-rich repeats have been shown to form G-quadruplex (G4) structures, which are crucial for modulating telomere functions. Surprisingly, even though plant telomeres are essential for plant growth, development, and environmental adaptions, only few reports exist on plant telomeric G4 DNA (pTG4). Here, using bulk and single-molecule assays, including CD spectroscopy, and single-molecule FRET approaches, we comprehensively characterized the structure and dynamics of a typical plant telomeric sequence, d[GGG(TTTAGGG)3]. We found that this sequence can fold into mixed G4s in potassium, including parallel and antiparallel structures. We also directly detected intermediate dynamic transitions, including G-hairpin, parallel G-triplex, and antiparallel G-triplex structures. Moreover, we observed that pTG4 is unfolded by the AtRecQ2 helicase but not by AtRecQ3. The results of our work shed light on our understanding about the existence, topological structures, stability, intermediates, unwinding, and functions of pTG4.

Haploinsufficiency of Meis homeobox 2 (MEIS2), encoding a transcriptional regulator, is associated with human cleft palate, and Meis2 inactivation leads to abnormal palate development in mice, implicating MEIS2 functions in palate development. However, its functional mechanisms remain unknown. Here we observed widespread MEIS2 expression in the developing palate in mice. Wnt1Cre-mediated Meis2 inactivation in cranial neural crest cells led to a secondary palate cleft. Importantly, about half of the Wnt1Cre;Meis2f/f mice exhibited a submucous cleft, providing a model for studying palatal bone formation and patterning. Consistent with complete absence of palatal bones, the results from integrative analyses of MEIS2 by ChIP sequencing, RNA-Seq, and an assay for transposase-accessible chromatin sequencing identified key osteogenic genes regulated directly by MEIS2, indicating that it plays a fundamental role in palatal osteogenesis. De novo motif analysis uncovered that the MEIS2-bound regions are highly enriched in binding motifs for several key osteogenic transcription factors, particularly short stature homeobox 2 (SHOX2). Comparative ChIP sequencing analyses revealed genome-wide co-occupancy of MEIS2 and SHOX2 in addition to their colocalization in the developing palate and physical interaction, suggesting that SHOX2 and MEIS2 functionally interact. However, although SHOX2 was required for proper palatal bone formation and was a direct downstream target of MEIS2, Shox2 overexpression failed to rescue the palatal bone defects in a Meis2-mutant background. These results, together with the fact that Meis2 expression is associated with high osteogenic potential and required for chromatin accessibility of osteogenic genes, support a vital function of MEIS2 in setting up a ground state for palatal osteogenesis.

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle—when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

VOLUME 294 (2019) PAGES 2555–2568Due to publisher error, “150 l/mm” was changed to “150 liters/mm” in the second paragraph of the “Vibrational spectroscopy of samples” section under “Experimental Procedures.” The correct phrase should be “150 l/mm.”

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.

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

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.

The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the SHP2 gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

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.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell–engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.