MYC translocations, a hallmark of Burkitt lymphoma, occur in 5-15% of diffuse large B-cell lymphoma, and have a negative prognostic impact. Numerical aberrations of MYC have also been detected in these patients, but their incidence and prognostic role are still controversial. We analyzed the clinical impact of MYC increased copy number on 385 patients with diffuse large B-cell lymphoma screened at diagnosis for MYC, BCL2, and BCL6 rearrangements. We enumerated the number of MYC copies, defining as amplified those cases with an uncountable number of extra-copies. The prevalence of MYC translocation, increased copy number and amplification was 8.8%, 15%, and 1%, respectively. Patients with 3 or 4 gene copies, accounting for more than 60% of patients with MYC copy number changes, had a more favorable outcome compared to patients with >4 copies or translocation of MYC, and were not influenced by the type of treatment received as first-line. Stratification according to the number of MYC extra-copies showed a negative correlation between an increasing number of copies and survival. Patients with >7 copies or the amplification of MYC had the poorest prognosis. Patients with >4 copies of MYC showed a similar, trending towards worse prognosis compared to patients with MYC translocation. The survival of patients with >4 copies, translocation or amplification of MYC seemed to be superior if intensive treatments were used. Our study underlines the importance of fluorescence in situ hybridization testing at diagnosis of diffuse large B-cell lymphoma to detect the rather frequent and clinically significant numerical aberrations of MYC.

B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.

Despite substantial progress in treatment of T-cell acute lymphoblastic leukemia (T-ALL), mortality remains relatively high, mainly due to primary or acquired resistance to chemotherapy. Further improvements in survival demand better understanding of T-ALL biology and development of new therapeutic strategies. The Notch pathway has been involved in the pathogenesis of this disease and various therapeutic strategies are currently under development, including selective targeting of NOTCH receptors by inhibitory antibodies. We previously demonstrated that the NOTCH1-specific neutralizing antibody OMP52M51 prolongs survival in TALL patient-derived xenografts bearing NOTCH1/FBW7 mutations. However, acquired resistance to OMP52M51 eventually developed and we used patient-derived xenografts models to investigate this phenomenon. Multi-level molecular characterization of T-ALL cells resistant to NOTCH1 blockade and serial transplantation experiments uncovered heterogeneous types of resistance, not previously reported with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired NOTCH1 mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was accompanied by modulation of lipid metabolism and reduced surface expression of NOTCH1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade NOTCH1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors.

Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML.

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL⁺ cells.

Although highly effective, BCR-ABL1 tyrosine kinase inhibitors do not target chronic myeloid leukemia (CML) stem cells. Most patients relapse upon tyrosine kinase inhibitor therapy cessation. We reported previously that combined BCR-ABL1 and BCL-2 inhibition synergistically targets CML stem/progenitor cells. p53 induces apoptosis mainly by modulating BCL-2 family proteins. Although infrequently mutated in CML, p53 is antagonized by MDM2, which is regulated by BCR-ABL1 signaling. We hypothesized that MDM2 inhibition could sensitize CML cells to tyrosine kinase inhibitors. Using an inducible transgenic Scl-tTa-BCR-ABL1 murine CML model, we found, by RT-PCR and CyTOF proteomics increased p53 signaling in CML bone marrow (BM) cells compared with controls in CD45⁺ and linage-SCA-1⁺C-KIT⁺ populations. CML BM cells were more sensitive to exogenous BH3 peptides than controls. Combined inhibition of BCR-ABL1 with imatinib and MDM2 with DS-5272 increased NOXA level, markedly reduced leukemic linage-SCA-1⁺C-KIT⁺ cells and hematopoiesis, decreased leukemia burden, significantly prolonged the survival of mice engrafted with BM cells from Scl-tTa-BCR-ABL1 mice, and significantly decreased CML stem cell frequency in secondary transplantations. Our results suggest that CML stem/progenitor cells have increased p53 signaling and a propensity for apoptosis. Combined MDM2 and BCR-ABL1 inhibition targets CML stem/progenitor cells and has the potential to improve cure rates for CML.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism <50% had hemolysis (reticulocytosis) and higher HbS than their donor. Four of them had donor chimerism <30%, including a patient with AA donor (hemoglobin >10 g/dL) and three with AS donors (hemoglobin <10 g/dL). However, only one vaso-occlusive crisis occurred with 68.7% HbS. Except in the patients with the lowest chimerism, the donor engraftment was lower for T cells than for the other lineages. In a context of mixed chimerism after hematopoietic stem cell transplantation for SCD, myeloid (rather than T cell) engraftment was the key efficacy criterion. Results show that myeloid chimerism as low as 30% was sufficient to prevent a vaso-occlusive crisis in transplants from an AA donor but not constantly from an AS donor. However, the correction of hemolysis requires higher donor chimerism levels (i.e. ≥50%) in both AA and AS recipients. In the future, this group of patients may need a different therapeutic approach.

In iron-depleted women without anemia, oral iron supplements induce an increase in serum hepcidin (SHep) that persists for 24 hours, decreasing iron absorption from supplements given later on the same or next day. Consequently, iron absorption from supplements is highest if iron is given on alternate days. Whether this dosing schedule is also beneficial in women with iron-deficiency anemia (IDA) given high-dose iron supplements is uncertain. The primary objective of this study was to assess whether, in women with IDA, alternate-day administration of 100 and 200 mg iron increases iron absorption compared to consecutive-day iron administration. Secondary objectives were to correlate iron absorption with SHep and iron status parameters. We performed a cross-over iron absorption study in women with IDA (n=19; median hemoglobin 11.5 mg/dL; mean serum ferritin 10 mg/L) who received either 100 or 200 mg iron as ferrous sulfate given at 8 AM on days 2, 3 and 5 labeled with stable iron isotopes 57Fe, 58Fe and 54Fe; after a 16-day incorporation period, the other labeled dose was given at 8 AM on days 23, 24 and 26 (days 2, 3 and 5 of the second period). Iron absorption on days 2 and 3 (consecutive) and day 5 (alternate) was assessed by measuring erythrocyte isotope incorporation. For both doses, SHep was higher on day 3 than on day 2 (P<0.001) or day 5 (P<0.01) with no significant difference between days 2 and 5. Similarly, for both doses, fractional iron absorption (FIA) on days 2 and 5 was 40-50% higher than on day 3 (P<0.001), while absorption on day 2 did not differ significantly from day 5. There was no significant difference in the incidence of gastrointestinal side effects comparing the two iron doses (P=0.105). Alternate day dosing of oral iron supplements in anemic women may be preferable because it sharply increases FIA. If needed, to provide the same total amount of iron with alternate day dosing, twice the daily target dose should be given on alternate days, as total iron absorption from a single dose of 200 mg given on alternate days was approximately twice that from 100 mg given on consecutive days (P<0.001). In IDA, even if hepatic hepcidin expression is strongly suppressed by iron deficiency and erythropoietic drive, the intake of oral iron supplements leads to an acute hepcidin increase for 24 hours. The study was funded by ETH Zürich, Switzerland. This study has been registered at www.clinicaltrials.gov as #NCT03623997.

This follow-up study of a randomized, prospective trial included 192 patients with newly diagnosed severe aplastic anemia receiving antithymoglobulin and cyclosporine, with or without granulocyte colony-stimulating factor (G-CSF). We aimed to evaluate the long-term effect of G-CSF on overall survival, event-free survival, probability of secondary myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), clinical paroxysmal nocturnal hemoglobinuria, relapse, avascular osteonecrosis and chronic kidney disease. The median follow-up was 11.7 years (95% CI, 10.9-12.5). The overall survival rate at 15 years was 57±12% in the group given G-CSF and 63±12% in the group not given G-CSF (P=0.92); the corresponding event-free survival rates were 24±10% and 23±10%, respectively (P=0.36). In total, 9 patients developed MDS or AML, 10 only a clonal cytogenetic abnormality, 7 a solid cancer, 18 clinical paroxysmal nocturnal hemoglobinuria, 8 osteonecrosis, and 12 chronic kidney disease, without any difference between patients treated with or without G-CSF. The cumulative incidence of MDS, AML or isolated cytogenetic abnormality at 15 years was 8.5±3% for the G-CSF group and 8.2±3% for the non-G-CSF group (P=0.90). The cumulative incidence of any late event including myelodysplastic syndrome or acute myeloid leukemia, isolated cytogenetic abnormalities, solid cancer, clinical paroxysmal nocturnal hemoglobinuria, aseptic osteonecrosis, chronic kidney disease and relapse was 50±12% for the G-CSF group and 49±12% for the non-G-CSF group (P=0.65). Our results demonstrate that it is unlikely that G-CSF has an impact on the outcome of severe aplastic anemia; nevertheless, very late events are common and eventually affect the prognosis of these patients, irrespectively of their age at the time of immunosuppressive therapy (NCT01163942).

Highly conserved among species and expressed in various types of cells, numerous roles have been attributed to the cellular prion protein (PrPC). In hematopoiesis, PrPC regulates hematopoietic stem cell self-renewal but the mechanisms involved in this regulation are unknown. Here we show that PrPC regulates hematopoietic stem cell number during aging and their determination towards myeloid progenitors. Furthermore, PrPC protects myeloid progenitors against the cytotoxic effects of total body irradiation. This radioprotective effect was associated with increased cellular prion mRNA level and with stimulation of the DNA repair activity of the Apurinic/pyrimidinic endonuclease 1, a key enzyme of the base excision repair pathway. Altogether, these results show a previously unappreciated role of PrPC in adult hematopoiesis, and indicate that PrPC-mediated stimulation of BER activity might protect hematopoietic progenitors from the cytotoxic effects of total body irradiation.

Human bone marrow stromal cells (BMSC) are key elements of the hematopoietic environment and they play a central role in bone and bone marrow physiology. However, how key stromal cell functions are regulated is largely unknown. We analyzed the role of the immediate early response transcription factor EGR1 as key stromal cell regulator and found that EGR1 was highly expressed in prospectively-isolated primary BMSC, down-regulated upon culture, and low in non-colony-forming CD45^neg stromal cells. Furthermore, EGR1 expression was lower in proliferative regenerating adult and fetal primary cells compared to adult steady-state BMSC. Overexpression of EGR1 in stromal cells induced potent hematopoietic stroma support as indicated by an increased production of transplantable CD34⁺CD90⁺ hematopoietic stem cells in expansion co-cultures. The improvement in bone marrow stroma support function was mediated by increased expression of hematopoietic supporting genes, such as VCAM1 and CCL28. Furthermore, EGR1 overexpression markedly decreased stromal cell proliferation whereas EGR1 knockdown caused the opposite effects. These findings thus show that EGR1 is a key stromal transcription factor with a dual role in regulating proliferation and hematopoietic stroma support function that is controlling a genetic program to co-ordinate the specific functions of BMSC in their different biological contexts.

Identifying gene variants causing monogenic diabetes (MD) increases understanding of disease etiology and allows for implementation of precision therapy to improve metabolic control and quality of life. Here, we aimed to assess the prevalence of MD in youth with diabetes in Lithuania, uncover potential diabetes-related gene variants, and prospectively introduce precision treatment. First, we assessed all pediatric and most young-adult patients with diabetes in Lithuania (n = 1,209) for diabetes-related autoimmune antibodies. We then screened all antibody-negative patients (n = 153) using targeted high-throughput sequencing of >300 potential candidate genes. In this group, 40.7% had MD, with the highest percentage (100%) in infants (diagnosis at ages 0–12 months), followed by those diagnosed at ages >1–18 years (40.3%) and >18–25 years (22.2%). The overall prevalence of MD in youth with diabetes in Lithuania was 3.5% (1.9% for GCK diabetes, 0.7% for HNF1A, 0.2% for HNF4A and ABCC8, 0.3% for KCNJ11, and 0.1% for INS). Furthermore, we identified likely pathogenic variants in 11 additional genes. Microvascular complications were present in 26% of those with MD. Prospective treatment change was successful in >50% of eligible candidates, with C-peptide >252 pmol/L emerging as the best prognostic factor.

Impaired baroreflex sensitivity (BRS) predicts cardiovascular mortality and is prevalent in long-term diabetes. We determined spontaneous BRS in patients with recent-onset diabetes and its temporal sequence over 5 years by recording beat-to-beat blood pressure and R-R intervals over 10 min. Four time domain and four frequency domain BRS indices were computed in participants from the German Diabetes Study baseline cohort with recent-onset type 1/type 2 diabetes (n = 206/381) and age-matched glucose-tolerant control subjects (control 1/control 2: n = 65/83) and subsets of consecutive participants with type 1/type 2 diabetes who reached the 5-year follow-up (n = 84/137). Insulin sensitivity (M-value) was determined using a hyperinsulinemic-euglycemic clamp. After appropriate adjustment, three frequency domain BRS indices were reduced in type 2 diabetes compared with control 2 and were positively associated with the M-value and inversely associated with fasting glucose and HbA_1c (P < 0.05), whereas BRS was preserved in type 1 diabetes. After 5 years, a decrease in one and four BRS indices was observed in patients with type 1 and type 2 diabetes, respectively (P < 0.05), which was explained by the physiologic age-dependent decline. Unlike patients with well-controlled recent-onset type 1 diabetes, those with type 2 diabetes show early baroreflex dysfunction, likely due to insulin resistance and hyperglycemia, albeit without progression over 5 years.

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet β-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3⁺ Treg-cell expansion in the spleen of wild-type mice but not in Gitr^–/– mice. G3C/cps also induced the expansion of nonconventional Cd4⁺Cd25^–/lowFoxp3^lowGitr^int/high (GITR single-positive [sp]) Tregs. Both Cd4⁺Cd25⁺Gitr^highFoxp3⁺ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene–induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in β-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.

Genes of the Notch signaling pathway are expressed in different cell types and organs at different time points during embryonic development and adulthood. The Notch ligand Delta-like 1 (DLL1) controls the decision between endocrine and exocrine fates of multipotent progenitors in the developing pancreas, and loss of Dll1 leads to premature endocrine differentiation. However, the role of Delta-Notch signaling in adult tissue homeostasis is not well understood. Here, we describe the spatial expression pattern of Notch pathway components in adult murine pancreatic islets and show that DLL1 and DLL4 are specifically expressed in β-cells, whereas JAGGED1 is expressed in α-cells. We show that mice lacking both DLL1 and DLL4 in adult β-cells display improved glucose tolerance, increased glucose-stimulated insulin secretion, and hyperglucagonemia. In contrast, overexpression of the intracellular domain of DLL1 in adult murine pancreatic β-cells results in impaired glucose tolerance and reduced insulin secretion, both in vitro and in vivo. These results suggest that Notch ligands play specific roles in the adult pancreas and highlight a novel function of the Delta/Notch pathway in β-cell insulin secretion.

An aging global population combined with sedentary lifestyles and unhealthy diets has contributed to an increasing incidence of obesity and type 2 diabetes. These metabolic disorders are associated with perturbations to nitric oxide (NO) signaling and impaired glucose metabolism. Dietary inorganic nitrate, found in high concentration in green leafy vegetables, can be converted to NO in vivo and demonstrates antidiabetic and antiobesity properties in rodents. Alongside tissues including skeletal muscle and liver, white adipose tissue is also an important physiological site of glucose disposal. However, the distinct molecular mechanisms governing the effect of nitrate on adipose tissue glucose metabolism and the contribution of this tissue to the glucose-tolerant phenotype remain to be determined. Using a metabolomic and stable-isotope labeling approach, combined with transcriptional analysis, we found that nitrate increases glucose uptake and oxidative catabolism in primary adipocytes and white adipose tissue of nitrate-treated rats. Mechanistically, we determined that nitrate induces these phenotypic changes in primary adipocytes through the xanthine oxidoreductase–catalyzed reduction of nitrate to NO and independently of peroxisome proliferator–activated receptor-α. The nitrate-mediated enhancement of glucose uptake and catabolism in white adipose tissue may be a key contributor to the antidiabetic effects of this anion.

Glucagon is classically described as a counterregulatory hormone that plays an essential role in the protection against hypoglycemia. In addition to its role in the regulation of glucose metabolism, glucagon has been described to promote ketosis in the fasted state. Sodium–glucose cotransporter 2 inhibitors (SGLT2i) are a new class of glucose-lowering drugs that act primarily in the kidney, but some reports have described direct effects of SGLT2i on α-cells to stimulate glucagon secretion. Interestingly, SGLT2 inhibition also results in increased endogenous glucose production and ketone production, features common to glucagon action. Here, we directly test the ketogenic role of glucagon in mice, demonstrating that neither fasting- nor SGLT2i-induced ketosis is altered by interruption of glucagon signaling. Moreover, any effect of glucagon to stimulate ketogenesis is severely limited by its insulinotropic actions. Collectively, our data suggest that fasting-associated ketosis and the ketogenic effects of SGLT2 inhibitors occur almost entirely independent of glucagon.

Accumulation of lipid in skeletal muscle is thought to be related to the development of insulin resistance and type 2 diabetes. Initial work in this area focused on accumulation of intramuscular triglyceride; however, bioactive lipids such as diacylglycerols and sphingolipids are now thought to play an important role. Specific species of these lipids appear to be more negative toward insulin sensitivity than others. Adding another layer of complexity, localization of lipids within the cell appears to influence the relationship between these lipids and insulin sensitivity. This article summarizes how accumulation of total lipids, specific lipid species, and localization of lipids influence insulin sensitivity in humans. We then focus on how these aspects of muscle lipids are impacted by acute and chronic aerobic and resistance exercise training. By understanding how exercise alters specific species and localization of lipids, it may be possible to uncover specific lipids that most heavily impact insulin sensitivity.

Insulin resistance is an underappreciated facet of type 1 diabetes that occurs with remarkable consistency and considerable magnitude. Although therapeutic innovations are continuing to normalize dysglycemia, a sizable body of data suggests a second metabolic abnormality—iatrogenic hyperinsulinemia—principally drives insulin resistance and its consequences in this population and has not been addressed. We review this evidence to show that injecting insulin into the peripheral circulation bypasses first-pass hepatic insulin clearance, which leads to the unintended metabolic consequence of whole-body insulin resistance. We propose restructuring insulin therapy to restore the physiological insulin balance between the hepatic portal and peripheral circulations and thereby avoid the complications of life-long insulin resistance. As technology rapidly advances and our ability to ensure euglycemia improves, iatrogenic insulin resistance will become the final barrier to overcome to restore normal physiology, health, and life in type 1 diabetes.

Advances in small RNA sequencing have revealed the enormous diversity of small noncoding RNA (sRNA) classes in mammalian cells. At this point, most investigators in diabetes are aware of the success of microRNA (miRNA) research and appreciate the importance of posttranscriptional gene regulation in glycemic control. Nevertheless, miRNAs are just one of multiple classes of sRNAs and likely represent only a minor fraction of sRNA sequences in a given cell. Despite the widespread appreciation of sRNAs, very little research into non-miRNA sRNA function has been completed, likely due to some major barriers that present unique challenges for study. To emphasize the importance of sRNA research in cardiometabolic diseases, we highlight the success of miRNAs and competitive endogenous RNAs in cholesterol and glucose metabolism. Moreover, we argue that sequencing studies have demonstrated that miRNAs are just the tip of the iceberg for sRNAs. We are likely standing at the precipice of immense discovery for novel sRNA-mediated gene regulation in cardiometabolic diseases. To realize this potential, we must first address critical barriers with an open mind and refrain from viewing non-miRNA sRNA function through the lens of miRNAs, as they likely have their own set of distinct regulatory factors and functional mechanisms.

Nayeli G. Reyes-Nava, Hung-Chun Yu, Curtis R. Coughlin II, Tamim H. Shaikh, and Anita M. Quintana

We used whole-exome sequencing (WES) to determine the genetic etiology of a patient with a multi-system disorder characterized by a seizure phenotype. WES identified a heterozygous de novo missense mutation in the GABRA1 gene (c.875C>T). GABRA1 encodes the alpha subunit of the gamma-aminobutyric acid receptor A (GABA_AR). The GABA_AR is a ligand gated ion channel that mediates the fast inhibitory signals of the nervous system, and mutations in the subunits that compose the GABA_AR have been previously associated with human disease. To understand the mechanisms by which GABRA1 regulates brain development, we developed a zebrafish model of gabra1 deficiency. gabra1 expression is restricted to the nervous system and behavioral analysis of morpholino injected larvae suggests that the knockdown of gabra1 results in hypoactivity and defects in the expression of other subunits of the GABA_AR. Expression of the human GABRA1 protein in morphants partially restored the hypomotility phenotype. In contrast, the expression of the c.875C>T variant did not restore these behavioral deficits. Collectively, these results represent a functional approach to understand the mechanisms by which loss-of-function alleles cause disease.

Chalongrat Noree and Naraporn Sirinonthanawech

Previously, we have developed an extramitochondrial assembly system, where mitochondrial targeting signal (MTS) can be removed from a given mitochondrial enzyme, which could be used to characterize the regulatory factors involved in enzyme assembly/disassembly in vivo. Here, we demonstrate that addition of exogenous acetaldehyde can quickly induce the supramolecular assembly of MTS-deleted aldehyde dehydrogenase Ald4p in yeast cytoplasm. Also, by using PCR-based modification of the yeast genome, cytoplasmically targeted Ald4p cannot polymerize into long filaments when key functional amino acid residues are substituted, as shown by N192D, S269A, E290K and C324A mutations. This study has confirmed that extramitochondrial assembly could be a powerful external system for studying mitochondrial enzyme assembly, and its regulatory factors outside the mitochondria. In addition, we propose that mitochondrial enzyme assembly/disassembly is coupled to the regulation of a given mitochondrial enzyme activity.

Yuta Sakae, Akira Oikawa, Yuki Sugiura, Masatoshi Mita, Shuhei Nakamura, Toshiya Nishimura, Makoto Suematsu, and Minoru Tanaka

The teleost fish, medaka (Oryzias latipes), employs the XX/XY genetic sex determination system. We show here that the phenotypic sex of medaka is affected by changes in lipid metabolism. Medaka larvae subjected to 5 days of starvation underwent female-to-male sex reversal. Metabolomic and RT-qPCR analyses indicated that pantothenate metabolism was suppressed by starvation. Consistently, inhibiting the pantothenate metabolic pathway caused sex reversal. The final metabolite in this pathway is coenzyme A, an essential factor for lipogenesis. Inhibiting fatty acid synthesis, the first step of lipogenesis, also caused sex reversal. The expression of dmrt1, a critical gene for male development, was suppressed by starvation, and a dmrt1 (13) mutant did not show sex reversal under starvation. Collectively, these results indicate that fatty acid synthesis is involved in female-to-male sex reversal through ectopic expression of male gene dmrt1 under starvation.

Manoella Gemaque Cavalcante, Cleusa Yoshiko Nagamachi, Julio Cesar Pieczarka, and Renata Coelho Rodrigues Noronha

Eukaryotic genomes exhibit substantial accumulation of repetitive DNA sequences. These sequences can participate in chromosomal reorganization events and undergo molecular cooption to interfere with the function and evolution of genomes. In turtles, repetitive DNA sequences appear to be accumulated at probable break points and may participate in events such as non-homologous recombination and chromosomal rearrangements. In this study, repeated sequences of 5S rDNA, U2 snRNA and Tc1/Mariner transposons were amplified from the genomes of the turtles, Podocnemis expansa and Podocnemis unifilis, and mapped by fluorescence in situ hybridization. Our data confirm the 2n=28 chromosomes for these species (the second lowest 2n in the order Testudines). We observe high conservation of the co-located 5S rDNA and U2 snRNA genes on a small chromosome pair (pair 13), and surmise that this represents the ancestral condition. Our analysis reveals a wide distribution of the Tc1/Mariner transposons and we discuss how the mobility of these transposons can act on karyotypic reorganization events (contributing to the 2n decrease of those species). Our data add new information for the order Testudines and provide important insights into the dynamics and organization of these sequences in the chelonian genomes.

Yan Xing, Hongling Wei, Xiumei Xiao, Zekun Chen, Hui Liu, Xiaomei Tong, and Wei Zhou

Infants with intrauterine growth retardation (IUGR) have a high risk of developing bronchial asthma in childhood, but the underlying mechanisms remain unclear. This study aimed to disclose the role of vascular non-inflammatory molecule 1 (vannin-1, encoded by the Vnn1 gene) and its downstream signaling in IUGR asthmatic mice induced by ovalbumin. Significant histological alterations and an increase of vannin-1 expression were revealed in IUGR asthmatic mice, accompanied by elevated methylation of Vnn1 promoter regions. In IUGR asthmatic mice, we also found (i) a direct binding of HNF4α and PGC1α to Vnn1 promoter by ChIP assay; (ii) a direct interaction of HNF4α with PGC1α; (iii) upregulation of phospho-PI3K p85/p55 and phospho-Akt^Ser473 and downregulation of phospho-PTENT^yr366, and (iv) an increase in nuclear NFB p65 and a decrease in cytosolic IB-α. In primary cultured bronchial epithelial cells derived from the IUGR asthmatic mice, knockdown of Vnn1 prevented upregulation of phospho-Akt^Ser473 and an increase of reactive oxygen species (ROS) and TGF-β production. Taken together, we demonstrate that elevated vannin-1 activates the PI3K/Akt/NFB signaling pathway, leading to ROS and inflammation reactions responsible for asthma occurrence in IUGR individuals. We also disclose that interaction of PGC1α and HNF4α promotes methylation of Vnn1 promoter regions and then upregulates vannin-1 expression.

Yu Liu, Shuhua Qi, Fridtjof Thomas, Brittany L. Correia, Angela P. Taylor, Roy V. Sillitoe, and Detlef H. Heck

Respiration is controlled by central pattern generating circuits in the brain stem, whose activity can be modulated by inputs from other brain areas to adapt respiration to autonomic and behavioral demands. The cerebellum is known to be part of the neuronal circuitry activated during respiratory challenges, such as hunger for air, but has not been found to be involved in the control of spontaneous, unobstructed breathing (eupnea). Here we applied a measure of intrinsic rhythmicity, the CV2, which evaluates the similarity of subsequent intervals and is thus sensitive to changes in rhythmicity at the temporal resolution of individual respiratory intervals. The variability of intrinsic respiratory rhythmicity was reduced in a mouse model of cerebellar ataxia compared to their healthy littermates. Irrespective of that difference, the average respiratory rate and the average coefficient of variation (CV) were comparable between healthy and ataxic mice. We argue that these findings are consistent with a proposed role of the cerebellum in modulating the duration of individual respiratory intervals, which could serve the purpose of coordinating respiration with other rhythmic orofacial movements, such as fluid licking and swallowing.

Soojeong Kim and Isabel K. Darcy

An experimental technique called difference topology combined with the mathematics of tangle analysis has been used to unveil the structure of DNA bound by the Mu transpososome. However, difference topology experiments can be difficult and time consuming. We discuss a modification that greatly simplifies this experimental technique. This simple experiment involves using a topoisomerase to trap DNA crossings bound by a protein complex and then running a gel to determine the crossing number of the knotted product(s). We develop the mathematics needed to analyze the results and apply these results to model the topology of DNA bound by 13S condensin and by the condensin MukB.

Sonu Shrestha Baral, Molly E. Lieux, and Patrick J. DiMario

Different stem cells or progenitor cells display variable threshold requirements for functional ribosomes. This is particularly true for several human ribosomopathies in which select embryonic neural crest cells or adult bone marrow stem cells, but not others, show lethality due to failures in ribosome biogenesis or function (now known as nucleolar stress). To determine if various Drosophila neuroblasts display differential sensitivities to nucleolar stress, we used CRISPR-Cas9 to disrupt the Nopp140 gene that encodes two splice variant ribosome biogenesis factors (RBFs). Disruption of Nopp140 induced nucleolar stress that arrested larvae in the second instar stage. While the majority of larval neuroblasts arrested development, the mushroom body (MB) neuroblasts continued to proliferate as shown by their maintenance of deadpan, a neuroblast-specific transcription factor, and by their continued EdU incorporation. MB neuroblasts in wild-type larvae appeared to contain more fibrillarin and Nopp140 in their nucleoli as compared to other neuroblasts, indicating that MB neuroblasts stockpile RBFs as they proliferate in late embryogenesis while other neuroblasts normally enter quiescence. A greater abundance of Nopp140 encoded by maternal transcripts in Nopp140-/- MB neuroblasts of 1­­­–2-day-old larvae likely rendered these cells more resilient to nucleolar stress.

This article has an associated First Person interview with the first author of the paper.

BackgroundSelf-harm is a serious risk factor for suicide, a major public health concern, and a significant burden on the NHS. Rates of self-harm presentation in primary care are rising and GPs interact with patients both before and after they have self-harmed. There is significant public and political interest in reducing rates of self-harm, but there has been no robust synthesis of the existing literature on the role of GPs in the management of patients who self-harm.AimThis study aimed to explore the role of the GP in the management of patients with self-harm behaviour.Design and settingA systematic review and narrative synthesis of primary care literature.MethodThis systematic review was conducted and is reported in line with PRISMA guidance. Electronic databases systematically searched were MEDLINE, PsycINFO, EMBASE, CINAHL, Web of Science, and AMED. Two independent reviewers conducted study screening and selection, data extraction, and quality appraisal of all included studies. Thematic analysis was conducted.ResultsFrom 6976 unique citations, 12 studies met eligibility criteria and were included. These 12 studies, published from 1997–2016, of 789 GPs/family medicine physicians from Europe, the US, and Australia were of good methodological quality. Five themes were identified for facilitating GP management of self-harm: GP training, improved communication, service provision, clinical guidelines, and young people. Four barriers for GP management of self-harm were identified: assessment, service provision, local, and systemic factors.ConclusionGPs recognise self-harm as a serious risk factor for suicide, but some feel unprepared for managing self-harm. The role of the GP is multidimensional and includes frontline assessment and treatment, referral to specialist care, and the provision of ongoing support.

BackgroundAutistic people are at increased risk of developing mental health problems. To reduce the negative impact of living with autism in a non-autistic world, efforts to improve take-up and access to care, and support in early years, which will typically start with a GP appointment, must be grounded in the accounts of autistic young adults.AimTo explore how autistic young adults understand and manage mental health problems; and to consider help seeking as a focus.Design and settingA cross-sectional, qualitative study. Autistic participants were purposively selected to represent a range of mental health conditions including anxiety and depression. A subsample were recruited from a population cohort screened for autism in childhood. The study concerns access to primary care.MethodNineteen autistic young adults without learning disabilities, aged 23 or 24 years, were recruited. In-depth, semi-structured interviews explored how they understood and managed mental health problems. Data were analysed thematically.ResultsYoung adults preferred self-management strategies. Multiple factors contributed to a focus on self-management, including: beliefs about the aetiology of mental health difficulties and increased vulnerability with the context of a diagnosis of autism, knowledge of self-management, and a view that formal support was unavailable or inadequate. Families had limited awareness of professional support.ConclusionYoung autistic adults without learning disabilities, and their families, may hold erroneous beliefs about autism and mental health. This may affect help seeking and contribute to an exacerbation of symptoms. GPs need to be alert to the fact that autistic young adults in their care may be experiencing mental health difficulties but may not recognise them as such.

BackgroundA brief intervention whereby GPs opportunistically facilitate an NHS-funded referral to a weight loss programme is clinically and cost-effective.AimTo test the acceptability of a brief intervention and attendance at a weight loss programme when GPs facilitate a referral that requires patients to pay for the service.Design and settingAn observational study of the effect of a GP encouraging attendance at a weight loss programme requiring self-payment in the West Midlands from 16 October 2018 to 30 November 2018, to compare with a previous trial in England in which the service was NHS-funded.MethodSixty patients with obesity who consecutively attended primary care appointments received an opportunistic brief intervention by a GP to endorse and offer a referral to a weight loss programme at the patient’s own expense. Participants were randomised to GPs who either stated the weekly monetary cost of the programme (basic cost) or who compared the weekly cost to an everyday discretionary item (cost comparison). Participants were subsequently asked to report whether they had attended a weight loss programme.ResultsOverall, 47% of participants (n = 28) accepted the referral; 50% (n = 15) in the basic cost group and 43% (n = 13) in the cost comparison group. This was significantly less than in a previous study when the programme was NHS-funded (77%, n = 722/940; P<0.0001). Most participants reported the intervention to be helpful/very helpful and appropriate/very appropriate (78%, n = 46/59 and 85%, n = 50/59, respectively) but scores were significantly lower than when the programme was NHS-funded (92% n = 851/922 and 88% n = 813/922, respectively; P = 0.004). One person (2%) attended the weight loss programme, which is significantly lower than the 40% of participants who attended when the programme was NHS-funded (P<0.0001).ConclusionGP referral to a weight loss programme that requires patients to pay rather than offering an NHS-funded programme is acceptable; however, it results in almost no attendance.

BackgroundAwareness of the importance of shared decision making (SDM) is widespread; however, little research has focused on discussions surrounding investigations, despite increasing laboratory testing in primary care.AimTo explore the discussion of blood tests in routine primary care consultations.Design and settingA secondary analysis of 50 video-recorded routine primary care consultations, linked surveys, and records data (all from the One in a Million [OiaM] archive). The consultations were taken by 22 GPs across 12 practices.MethodA coding scheme was developed, using qualitative content analysis, to explore discussion of blood tests in transcripts of recorded consultations. Codes focused on instigating testing, the extent of SDM, and how results were explained. Survey data were used to compare patients’ pre-visit expectations with consultation content. Medical records were reviewed to compare tests discussed with those ordered.ResultsIn 36 out of 50 consultations that discussed ordering blood tests, 11 patients (31%) hinted that they wanted a blood test; however, none asked explicitly. Only four patients (11%) were offered alternative options. In 29 cases (81%) the GP gave some explanation of the indication, but only in six cases (17%) were the limitations of testing explained. Only 10 out of 31 patients (32%) were informed about all blood tests ordered. Of the 23 out of 50 consultations in which results were conveyed, the GP gave no explanation of the results in six cases (26%). Thirteen patients (57%) were only informed of an assessment of the results (for example, ‘normal’), rather than the actual results.ConclusionA lack of information dissemination and SDM exists around ordering tests and conveying results. Promoting SDM could reduce unnecessary testing and improve patient-centred care.

BackgroundUrinary tract infections (UTIs) are one of the most common bacterial infections managed in general practice. Many women with symptoms of uncomplicated UTI may not benefit meaningfully from antibiotic treatment, but the evidence base is complex and there is no suitable shared decision-making resource to guide antibiotic treatment and symptomatic care for use in general practice consultations.AimTo develop an evidence-based, shared decision-making intervention leaflet to optimise management of uncomplicated UTI for women aged <65 years in the primary care setting.Design and settingQualitative telephone interviews with GPs and patient focus group interviews.MethodIn-depth interviews were conducted to explore how consultation discussions around diagnosis, antibiotic use, self-care, safety netting, and prevention of UTI could be improved. Interview schedules were based on the Theoretical Domains Framework.ResultsBarriers to an effective joint consultation and appropriate prescribing included: lack of GP time, misunderstanding of depth of knowledge and miscommunication between the patient and the GP, nature of the consults (such as telephone consultations), and a history of previous antibiotic therapy.ConclusionConsultation time pressures combined with late symptom presentation are a challenge for even the most experienced of GPs: however, it is clear that enhanced patient–clinician shared decision making is urgently required when it comes to UTIs. This communication should incorporate the provision of self-care, safety netting, and preventive advice to help guide patients when to consult. A shared decision-making information leaflet was iteratively co-produced with patients, clinicians, and researchers at Public Health England using study data.