Membrane lipids couple synaptotagmin to SNARE-mediated granule fusion in insulin-secreting cells.

Insulin secretion depends on the Ca2+-regulated fusion of granules with the plasma membrane. A recent model of Ca2+-triggered exocytosis in secretory cells proposes that lipids in the plasma membrane couple the calcium sensor Syt1 to the membrane fusion machinery (Kiessling et al., 2018). Specifically, Ca2+-mediated binding of Syt1's C2 domains to the cell membrane shifts the membrane-anchored SNARE syntaxin-1a to a more fusogenic conformation, straightening its juxtamembrane linker. To test this model in live cells and extend it to insulin secretion, we enriched INS1 cells with a panel of lipids with different acyl chain compositions. Fluorescence lifetime measurements demonstrate that cells with more disordered membranes show an increase in fusion efficiency, and vice versa. Experiments with granules purified from INS1 cells and recombinant SNARE proteins reconstituted in supported membranes confirmed that lipid acyl chain composition determines SNARE conformation and that lipid disordering correlates with increased fusion. Addition of Syt1's C2AB domains significantly decreased lipid order in target membranes and increased SNARE-mediated fusion probability. Strikingly, Syt's action on both fusion and lipid order could be partially bypassed by artificially increasing unsaturated phosphatidylserines in the target membrane. Thus, plasma membrane lipids actively participate in coupling Ca2+/synaptotagmin-sensing to the SNARE fusion machinery in cells.

Analogs of FTY720 inhibit TRPM7 but not S1PRs and exert multimodal anti-inflammatory effects.

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we test non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo, for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, though this likely occurrs through multiple molecular targets. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo. Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting which demonstrate potent, polymodal anti-inflammatory activities ex vivo and in vivo.

Novel TRPM7 inhibitors with potent anti-inflammatory effects in vivo.

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine 1-phosphate (S1P) receptors. In this study, we tested non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo , for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, an effect that is predominantly but not solely mediated by TRPM7. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo . Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting. These inhibitors exhibit potent anti-inflammatory properties that are mediated by TRPM7 and likely other molecular targets that remain to be identified.

Adherence to CONSORT Guidelines and Reporting of the Determinants of External Validity in Clinical Oncology Randomized Controlled Trials: A Review of Trials Published in Four Major Journals between 2013 and 2015.

Our primary objective was to determine the proportion of trials that report the number of patients assessed for eligibility before randomization. We performed the systematic retrieval and analysis of all phase II, III, and IV RCTs published between 2013 and 2015 in four high-impact-factor journals in the field of clinical oncology. Among 456 RCTs reviewed, 236 trials (51.8%) reported the number of patients assessed for eligibility. Among the 236 trials that reported the entire enrollment process, the reasons for patient exclusion could be found in 184 trials (78%). A flow diagram was presented in 452 trials (99.1%), and 98 trials (21.5%) included a discussion on generalizability. Reporting the parameters of external validity in medical oncology RCTs is challenging. Improving adherence to the 2010 CONSORT guidelines concerning the enrollment process could help clinicians and health policymakers establish to whom trial results apply.

CAR T-Cells for the Treatment of Refractory or Relapsed Large B-Cell Lymphoma: A Single-Center Retrospective Canadian Study.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells are an important new third-line treatment option for large B-cell lymphoma (LBCL). The objective response rates in pivotal early phase clinical trials with CAR T-cells were very promising. The objective of this study was to describe the efficacy results obtained with CAR T-cells infusions in our institution and to compare the toxicities of our cohort with those of pivotal trials and studies conducted in a real-life setting. PATIENTS AND METHODS: Efficacy and safety data were retrospectively collected from 25 patients with LBCL treated with CAR T-cells therapy at CHU de Québec-Université Laval. A literature search was then performed to identify other efficacy or safety data from a real-life setting. RESULTS: At 3 months post infusion, the objective response rate (ORR) in our population with tisagenlecleucel and axicabtagene-ciloleucel were 20% and 47%, respectively. Bulky disease was the only negative predictor of poor response at 3 months (0% vs. 53%, P = .03). Bulky disease was associated with a median PFS of 2 months compared to 5 months for non-bulky disease (P = .0009). Grade ≥ 3 hematological toxicities were greater in patients treated with axi-cel (60% vs. 20%, P = .048), without bone marrow involvement (55% vs. 0%, P =.046), without stage IV disease (72% vs. 21%, P =.02), with refractory disease (67% vs. 10%, P =.01) or having been affected by cytokine release syndrome (58% vs. 0%, P =.02). CONCLUSION: The poor response rate at 3 months after infusion in our cohort was influenced mainly by bulky disease. Further studies are needed to better characterize the loss of efficacy of CAR T-cells because the majority of patients will relapse over time.

An Analysis of Formal Patient Complaints, Risk, and Malpractice Events Involving Orthopedic Trauma Surgeons During a 10-Year Period.

Formal patient complaints and malpractice events involving orthopedic trauma surgeons (OTSs) can have substantial career implications. Our purpose was to analyze formal patient complaints, risk events, and malpractice events against OTSs during a 10-year period. We reviewed all formal patient complaints within our institution's patient advocacy database involving 9 fellowship-trained OTSs throughout a decade. Complaints were categorized using the Patient Complaint Analysis System. Potential risk and malpractice events involving the OTSs were recorded. A control group of all patients seen by the surgeons during the study period was created. Demographics between patients with complaints and the control group were analyzed, as were malpractice, risk, and complaint rates between the surgeons. Of 33,770 patients, 136 filed a formal complaint (0.40%). There were 29 malpractice claims and 2 malpractice lawsuits. The care and treatment domain accounted for the highest percentage of complaints (36%), followed by the access and availability domain (26%). Results of the logistic regression analysis indicated that private insurance (odds ratio, 1.58) and operative treatment (odds ratio, 3.65) were significantly associated with complaints. Despite statistically significant differences in the rates of complaint and risk events between surgeons, malpractice events did not differ. The rate of patient complaints within a large orthopedic trauma practice during a 10-year period was 0.40%. Patients with private insurance and those treated operatively were more likely to file a complaint. Whereas complaint rates among surgeons varied, there was no significant difference in the rate of malpractice events. Understanding patient complaint rates and categorizations may allow surgeons to target areas for improvement. [Orthopedics. 2023;46(2):121-127.].

Elucidating the Role of Optical Activity of Polymers in Protein-Polymer Interactions.

Proteins are biomolecules with potential applications in agriculture, food sciences, pharmaceutics, biotechnology, and drug delivery. Interactions of hydrophilic and biocompatible polymers with proteins may impart proteolytic stability, improving the therapeutic effects of biomolecules and also acting as excipients for the prolonged storage of proteins under harsh conditions. The interactions of hydrophilic and stealth polymers such as poly(ethylene glycol), poly(trehalose), and zwitterionic polymers with various proteins are well studied. This study evaluates the molecular interactions of hydrophilic and optically active poly(vitamin B5 analogous methacrylamide) (poly(B5AMA)) with model proteins by fluorescence spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and circular dichroism (CD) spectroscopy analysis. The optically active hydrophilic polymers prepared using chiral monomers of R-(+)- and S-(-)-B5AMA by the photo-iniferter reversible addition fragmentation chain transfer (RAFT) polymerization showed concentration-dependent weak interactions of the polymers with bovine serum albumin and lysozyme proteins. Poly(B5AMA) also exhibited a concentration-dependent protein stabilizing effect at elevated temperatures, and no effect of the stereoisomers of polymers on protein thermal stability was observed. NMR analysis, however, showed poly(B5AMA) stereoisomer-dependent changes in the secondary structure of proteins.

SARS-CoV-2 requires acidic pH to infect cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein-catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. Significance Statement: SARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

Efferocytosis requires periphagosomal Ca2+-signaling and TRPM7-mediated electrical activity.

Efficient clearance of apoptotic cells by phagocytosis, also known as efferocytosis, is fundamental to developmental biology, organ physiology, and immunology. Macrophages use multiple mechanisms to detect and engulf apoptotic cells, but the signaling pathways that regulate the digestion of the apoptotic cell cargo, such as the dynamic Ca2+ signals, are poorly understood. Using an siRNA screen, we identify TRPM7 as a Ca2+-conducting ion channel essential for phagosome maturation during efferocytosis. Trpm7-targeted macrophages fail to fully acidify or digest their phagosomal cargo in the absence of TRPM7. Through perforated patch electrophysiology, we demonstrate that TRPM7 mediates a pH-activated cationic current necessary to sustain phagosomal acidification. Using mice expressing a genetically-encoded Ca2+ sensor, we observe that phagosome maturation requires peri-phagosomal Ca2+-signals dependent on TRPM7. Overall, we reveal TRPM7 as a central regulator of phagosome maturation during macrophage efferocytosis.

SARS-CoV-2 requires acidic pH to infect cells.

SARS-CoV-2 cell entry starts with membrane attachment and ends with spike-protein (S) catalyzed membrane fusion depending on two cleavage steps, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time 3D single virion tracking, we show fusion and genome penetration requires virion exposure to an acidic milieu of pH 6.2-6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2 overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2 expressing cells in the acidic milieu of the nasal cavity. Significance Statement: Infection by SARS-CoV-2 depends upon the S large spike protein decorating the virions and is responsible for receptor engagement and subsequent fusion of viral and cellular membranes allowing release of virion contents into the cell. Using new single particle imaging tools, to visualize and track the successive steps from virion attachment to fusion, combined with chemical and genetic perturbations of the cells, we provide the first direct evidence for the cellular uptake routes of productive infection in multiple cell types and their dependence on proteolysis of S by cell surface or endosomal proteases. We show that fusion and content release always require the acidic environment from endosomes, preceded by liberation of the S1 fragment which depends on ACE2 receptor engagement. One sentence summary: Detailed molecular snapshots of the productive infectious entry pathway of SARS-CoV-2 into cells.

Putative neural consequences of captivity for elephants and cetaceans.

The present review assesses the potential neural impact of impoverished, captive environments on large-brained mammals, with a focus on elephants and cetaceans. These species share several characteristics, including being large, wide-ranging, long-lived, cognitively sophisticated, highly social, and large-brained mammals. Although the impact of the captive environment on physical and behavioral health has been well-documented, relatively little attention has been paid to the brain itself. Here, we explore the potential neural consequences of living in captive environments, with a focus on three levels: (1) The effects of environmental impoverishment/enrichment on the brain, emphasizing the negative neural consequences of the captive/impoverished environment; (2) the neural consequences of stress on the brain, with an emphasis on corticolimbic structures; and (3) the neural underpinnings of stereotypies, often observed in captive animals, underscoring dysregulation of the basal ganglia and associated circuitry. To this end, we provide a substantive hypothesis about the negative impact of captivity on the brains of large mammals (e.g., cetaceans and elephants) and how these neural consequences are related to documented evidence for compromised physical and psychological well-being.

Deacetylation as a receptor-regulated direct activation switch for pannexin channels.

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.

Hypoxia-inducible factor prolyl hydroxylase domain (PHD) inhibition after contusive spinal cord injury does not improve locomotor recovery.

Traumatic spinal cord injury (SCI) is a devastating neurological condition that involves both primary and secondary tissue loss. Various cytotoxic events including hypoxia, hemorrhage and blood lysis, bioenergetic failure, oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation contribute to secondary injury. The HIF prolyl hydroxylase domain (PHD/EGLN) family of proteins are iron-dependent, oxygen-sensing enzymes that regulate the stability of hypoxia inducible factor-1α (HIF-1α) and also mediate oxidative stress caused by free iron liberated from the lysis of blood. PHD inhibition improves outcome after experimental intracerebral hemorrhage (ICH) by reducing activating transcription factor 4 (ATF4)-driven neuronal death. As the ATF4-CHOP (CCAAT-enhancer-binding protein homologous protein) pathway plays a role in the pathogenesis of contusive SCI, we examined the effects of PHD inhibition in a mouse model of moderate T9 contusive SCI in which white matter damage is the primary driver of locomotor dysfunction. Pharmacological inhibition of PHDs using adaptaquin (AQ) moderately lowers acute induction of Atf4 and Chop mRNAs and prevents the acute decline of oligodendrocyte (OL) lineage mRNAs, but does not improve long-term recovery of hindlimb locomotion or increase chronic white matter sparing. Conditional genetic ablation of all three PHD isoenzymes in OLs did not affect Atf4, Chop or OL mRNAs expression levels, locomotor recovery, and white matter sparing after SCI. Hence, PHDs may not be suitable targets to improve outcomes in traumatic CNS pathologies that involve acute white matter injury.

Mitochondrial Ca2+ Signaling Is an Electrometabolic Switch to Fuel Phagosome Killing.

Phagocytes reallocate metabolic resources to kill engulfed pathogens, but the intracellular signals that rapidly switch the immunometabolic program necessary to fuel microbial killing are not understood. We report that macrophages use a fast two-step Ca2+ relay to meet the bioenergetic demands of phagosomal killing. Upon detection of a fungal pathogen, macrophages rapidly elevate cytosolic Ca2+ (phase 1), and by concurrently activating the mitochondrial Ca2+ (mCa2+) uniporter (MCU), they trigger a rapid influx of Ca2+ into the mitochondria (phase 2). mCa2+ signaling reprograms mitochondrial metabolism, at least in part, through the activation of pyruvate dehydrogenase (PDH). Deprived of mCa2+ signaling, Mcu-/- macrophages are deficient in phagosomal reactive oxygen species (ROS) production and defective at killing fungi. Mice lacking MCU in their myeloid cells are highly susceptible to disseminated candidiasis. In essence, this study reveals an elegant design principle that MCU-dependent Ca2+ signaling is an electrometabolic switch to fuel phagosome killing.

Distinct insulin granule subpopulations implicated in the secretory pathology of diabetes types 1 and 2.

Insulin secretion from ß-cells is reduced at the onset of type-1 and during type-2 diabetes. Although inflammation and metabolic dysfunction of ß-cells elicit secretory defects associated with type-1 or type-2 diabetes, accompanying changes to insulin granules have not been established. To address this, we performed detailed functional analyses of insulin granules purified from cells subjected to model treatments that mimic type-1 and type-2 diabetic conditions and discovered striking shifts in calcium affinities and fusion characteristics. We show that this behavior is correlated with two subpopulations of insulin granules whose relative abundance is differentially shifted depending on diabetic model condition. The two types of granules have different release characteristics, distinct lipid and protein compositions, and package different secretory contents alongside insulin. This complexity of ß-cell secretory physiology establishes a direct link between granule subpopulation and type of diabetes and leads to a revised model of secretory changes in the diabetogenic process.

Diabetes is a disease that occurs when sugar levels in the blood can no longer be controlled by a hormone called insulin. People with type 1 diabetes lose the ability to produce insulin after their immune system attacks the ß-cells in their pancreas that make this hormone. People with type 2 diabetes develop the disease when ß-cells become exhausted from increased insulin demand and stop producing insulin. ß-cells store insulin in small compartments called granules. When blood sugar levels rise, these granules fuse with the cell membrane allowing ß-cells to release large quantities of insulin at once. This fusion is disrupted early in type 1 diabetes, but later in type 2: the underlying causes of these disruptions are unclear. In the laboratory, signals that trigger inflammation and molecules called fatty acids can mimic type 1 or type 2 diabetes respectively when applied to insulin-producing cells. Kreutzberger, Kiessling et al. wanted to know whether pro-inflammatory molecules and fatty acids affect insulin granules differently at the molecular level. To do this, insulin-producing cells were grown in the lab and treated with either fatty acids or pro-inflammatory molecules. The insulin granules of these cells were then isolated. Next, the composition of the granules and how they fused to lab-made membranes that mimic the cell membrane was examined. The experiments revealed that healthy ß-cells have two types of granules, each with a different version of a protein called synaptotagmin. Cells treated with molecules mimicking type 1 diabetes lost granules with synaptotagmin-7, while granules with synaptotagmin-9 were lost in cells treated with fatty acids to imitate type 2 diabetes. Each type of granule responded differently to calcium levels in the cell and secreted different molecules, indicating that each elicits a different diabetic response in the body. These findings suggest that understanding how insulin granules are formed and regulated may help find treatments for type 1 and 2 diabetes, possibly leading to therapies that reverse the loss of different types of granules. Additionally, the molecules of these granules may also be used as markers to determine the stage of diabetes. More broadly, these results show how understanding how molecule release changes with disease in different cell types may help diagnose or stage a disease.

Morphometric analysis of the second through fifth metacarpal through posteroanterior X-Rays.

Over the past 10 years, metacarpal fractures have had an annual incidence of 13.6 per 10,000 individuals. Literature has not reviewed anatomical variations through radiographic imaging, which may play a role in reducing postoperative complications. The purpose of this study was to use radiographic imaging to provide a detailed anatomy of the second through fifth metacarpals. This retrospective study measured length, neck width, narrowest body width, and narrowest medullary canal width of the second through fifth metacarpals through the use of posteroanterior X-rays. Patients who were ≥18 years and received hand radiographs from January 2015 to July 2019 were included in this study. Those with acute injury or fracture of the metacarpal were excluded. Five hundred and seventy-two metacarpals were included in this study, with 143 metacarpals measured each for the second through fifth metacarpal. The second metacarpal had the largest measured length, neck width, and narrowest body width at 68.72, 12.34, and 8.74 mm, respectively. The fifth metacarpal had the greatest average medullary canal width at 4.15 mm. This is the largest study in literature to comprehensively examine the anatomical variation of the second through fifth metacarpals. The second metacarpal had greatest dimensions except for canal width, which was the fifth metacarpal. Men almost consistently had greater metacarpal size when compared to women, and age was associated with second and third metacarpal canal width. The increased knowledge of metacarpal anatomy may potentially lay the foundation of further improvement of metacarpal implants and potentially reduce postoperative complications. Clin. Anat., 33:1014-1018, 2020. © 2019 Wiley Periodicals, Inc.

Biased M1 receptor-positive allosteric modulators reveal role of phospholipase D in M1-dependent rodent cortical plasticity.

Highly selective, positive allosteric modulators (PAMs) of the M1 subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach to potentially improve cognitive function in patients suffering from Alzheimer's disease and schizophrenia. Discovery programs have produced a structurally diverse range of M1 receptor PAMs with distinct pharmacological properties, including different extents of agonist activity and differences in signal bias. This includes biased M1 receptor PAMs that can potentiate coupling of the receptor to activation of phospholipase C (PLC) but not phospholipase D (PLD). However, little is known about the role of PLD in M1 receptor signaling in native systems, and it is not clear whether biased M1 PAMs display differences in modulating M1-mediated responses in native tissue. Using PLD inhibitors and PLD knockout mice, we showed that PLD was necessary for the induction of M1-dependent long-term depression (LTD) in the prefrontal cortex (PFC). Furthermore, biased M1 PAMs that did not couple to PLD not only failed to potentiate orthosteric agonist-induced LTD but also blocked M1-dependent LTD in the PFC. In contrast, biased and nonbiased M1 PAMs acted similarly in potentiating M1-dependent electrophysiological responses that were PLD independent. These findings demonstrate that PLD plays a critical role in the ability of M1 PAMs to modulate certain central nervous system (CNS) functions and that biased M1 PAMs function differently in brain regions implicated in cognition.