Mechanism of hydrophobic gating in the acetylcholine receptor channel pore.

Neuromuscular acetylcholine receptors (AChRs) are hetero-pentameric, ligand-gated ion channels. The binding of the neurotransmitter acetylcholine (ACh) to two target sites promotes a global conformational change of the receptor that opens the channel and allows ion conduction through the channel pore. Here, by measuring free-energy changes from single-channel current recordings and using molecular dynamics simulations, we elucidate how a constricted hydrophobic region acts as a "gate" to regulate the channel opening in the pore of AChRs. Mutations of gate residues, including those implicated in congenital myasthenia syndrome, lower the permeation barrier of the channel substantially and increase the unliganded gating equilibrium constant (constitutive channel openings). Correlations between hydrophobicity and the observed free-energy changes, supported by calculations of water densities in the wild-type versus mutant channel pores, provide evidence for hydrophobic wetting-dewetting transition at the gate. The analysis of a coupled interaction network provides insight into the molecular mechanism of closed- versus open-state conformational changes at the gate. Studies of the transition state by "phi"(φ)-value analysis indicate that agonist binding serves to stabilize both the transition and the open state. Intersubunit interaction energy measurements and molecular dynamics simulations suggest that channel opening involves tilting of the pore-lining M2 helices, asymmetric outward rotation of amino acid side chains, and wetting transition of the gate region that lowers the barrier to ion permeation and stabilizes the channel open conformation. Our work provides new insight into the hydrophobic gate opening and shows why the gate mutations result in constitutive AChR channel activity.

Neural inference at the frontier of energy, space, and time.

Computing, since its inception, has been processor-centric, with memory separated from compute. Inspired by the organic brain and optimized for inorganic silicon, NorthPole is a neural inference architecture that blurs this boundary by eliminating off-chip memory, intertwining compute with memory on-chip, and appearing externally as an active memory chip. NorthPole is a low-precision, massively parallel, densely interconnected, energy-efficient, and spatial computing architecture with a co-optimized, high-utilization programming model. On the ResNet50 benchmark image classification network, relative to a graphics processing unit (GPU) that uses a comparable 12-nanometer technology process, NorthPole achieves a 25 times higher energy metric of frames per second (FPS) per watt, a 5 times higher space metric of FPS per transistor, and a 22 times lower time metric of latency. Similar results are reported for the Yolo-v4 detection network. NorthPole outperforms all prevalent architectures, even those that use more-advanced technology processes.

DMF-Net: a deep multi-level semantic fusion network for high-resolution chest CT and X-ray image de-noising.

Medical images such as CT and X-ray have been widely used for the detection of several chest infections and lung diseases. However, these images are susceptible to different types of noise, and it is hard to remove these noises due to their complex distribution. The presence of such noise significantly deteriorates the quality of the images and significantly affects the diagnosis performance. Hence, the design of an effective de-noising technique is highly essential to remove the noise from chest CT and X-ray images prior to further processing. Deep learning methods, mainly, CNN have shown tremendous progress on de-noising tasks. However, existing CNN based models estimate the noise from the final layers, which may not carry adequate details of the image. To tackle this issue, in this paper a deep multi-level semantic fusion network is proposed, called DMF-Net for the removal of noise from chest CT and X-ray images. The DMF-Net mainly comprises of a dilated convolutional feature extraction block, a cascaded feature learning block (CFLB) and a noise fusion block (NFB) followed by a prominent feature extraction block. The CFLB cascades the features from different levels (convolutional layers) which are later fed to NFB to attain correct noise prediction. Finally, the Prominent Feature Extraction Block(PFEB) produces the clean image. To validate the proposed de-noising technique, a separate and a mixed dataset containing high-resolution CT and X-ray images with specific and blind noise are used. Experimental results indicate the effectiveness of the DMF-Net compared to other state-of-the-art methods in the context of peak signal-to-noise ratio (PSNR) and structural similarity measurement (SSIM) while drastically cutting down on the processing power needed.

Bispidine as a Versatile Scaffold: From Topological Hosts to Transmembrane Transporters.

The development of designer topological structures is a synthetically challenging endeavor. We present herein bispidine as a platform for the design of molecules with various topologies and functions. The bispidine-based acyclic molecule, which shows intriguing S-shape topology, is discussed. Single-crystal X-ray diffraction studies revealed that this molecule exists in the solid state as two conformational enantiomers. In addition, bispidine-based designer macrocycles were synthesized and investigated for ionophoric properties. Patch clamp experiments revealed that these macrocycles transport both anions and cations non-specifically with at least tenfold higher chloride conductance over the cations under the given experimental conditions. Ultramicroscopy and single-crystal X-ray crystallographic studies indicated that the self-assembling macrocycle forms a tubular assembly. Our design highlights the use of unconventional dihydrogen interactions in nanotube fabrication.

Results of an open-label phase 1b study of the ERK inhibitor MK-8353 plus the MEK inhibitor selumetinib in patients with advanced or metastatic solid tumors.

AIM: We evaluated MK-8353 (small molecule inhibitor of extracellular signal-regulated kinase 1/2) plus selumetinib (mitogen-activated extracellular signal-regulated kinase 1/2 inhibitor) in patients with advanced solid tumors. METHODS: This phase 1b, open-label, dose-escalation study (NCT03745989) enrolled adults with histologically/cytologically documented, locally advanced/metastatic solid tumors. MK-8353/selumetinib dose combinations were intended to be investigated in sequence: 50/25, 100/50, 150/75, 200/75, 200/100, and 250/100. Each agent was administered orally BID 4 days on/3 days off in repeating cycles every 21 days. Primary objectives were safety and tolerability and to establish preliminary recommended phase 2 doses for combination therapy. RESULTS: Thirty patients were enrolled. Median (range) age was 61.5 (26-78) years and 93% had received previous cancer therapy. Among 28 patients in the dose-limiting toxicities [DLT]-evaluable population, 8 experienced DLTs: 1/11 (9%) in the MK-8353/selumetinib 100/50-mg dose level experienced a grade 3 DLT (urticaria), and 7/14 (50%) in the 150/75-mg dose level experienced grade 2/3 DLTs (n = 2 each of blurred vision, retinal detachment, vomiting; n = 1 each of diarrhea, macular edema, nausea, retinopathy). The DLT rate in the latter dose level exceeded the prespecified target DLT rate (~30%). Twenty-six patients (87%) experienced treatment-related adverse events (grade 3, 30%; no grade 4/5), most commonly diarrhea (67%), nausea (37%), and acneiform dermatitis (33%). Three patients (10%) experienced treatment-related adverse events leading to treatment discontinuation. Best response was stable disease in 14 patients (n = 10 with MK-8353/selumetinib 150/75 mg). CONCLUSION: MK-8353/selumetinib 50/25 mg and 100/50 mg had acceptable safety and tolerability, whereas 150/75 mg was not tolerable. No responses were observed.

Predictors of morbidity in revisional bariatric surgery and bariatric emergencies at an MBSAQIP-accredited community hospital.

INTRODUCTION: Bariatric surgery revisions and emergencies are associated with higher morbidity and mortality compared to primary bariatric surgery. No formal outcome benchmarks exist that distinguish MBSAQIP-accredited centers in the community from unaccredited institutions. METHODS: A retrospective chart review was conducted on 53 bariatric surgery revisions and 61 bariatric surgical emergencies by a single surgeon at a high-volume community hospital accredited program from 2018 to 2020. Primary outcomes were complications or deaths occurring within 30-days of the index procedure. Secondary outcomes included operative time, leaks, surgical site occurrences (SSOs), and deep surgical site infections. RESULTS: There were no significant differences in the demographic characteristics of the study groups. Mean operative time was significantly longer for revisions as compared to emergency operations (149.5 vs. 89.4 min). Emergencies had higher surgical site infection (5.7% vs. 21.3%, p < 0.05) and surgical site occurrence (SSO) (1.9% vs. 29.5%, p < 0.05) rates compared to revisions. Logistic regression analysis identified several factors to be predictive of increased risk of morbidity: pre-operative albumin < 3.5 g/dL (p < 0.05), recent bariatric procedure within the last 30 days (p < 0.05), prior revisional bariatric surgery (p < 0.05), prior duodenal switch (p < 0.05), and pre-operative COPD (p < 0.05). CONCLUSION: Bariatric surgery revisions and emergencies have similar morbidity and mortality, far exceeding those of the primary operation. Outcomes comparable to those reported by urban academic centers can be achieved in community hospital MBSAQIP-accredited centers.

[68Ga]Ga-DOTA-Siglec-9 Detects Pharmacodynamic Changes of FAP-Targeted IL2 Variant Immunotherapy in B16-FAP Melanoma Mice.

Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible adhesion molecule, which supports contact between leukocytes and inflamed endothelium. There is evidence that VAP-1 is involved in the recruitment of leukocytes to melanoma tumors. Interleukin-2 (IL-2)-based immunotherapy is an efficient therapy that promotes immune system activity against cancers but is associated with toxicity. In the present study, we evaluated the feasibility of PET/CT imaging using the radiotracer [68Ga]Ga-DOTA-Siglec-9, which is targeted to VAP-1, to monitor pharmacodynamic effects of a novel FAP-IL2v immunocytokine (a genetically engineered variant of IL-2 fused with fibroblast activation protein) in the B16-FAP melanoma model. At 9 days after the inoculation of B16-FAP melanoma cells, mice were studied with [68Ga]Ga-DOTA-Siglec-9 PET/CT as a baseline measurement. Immediately after baseline imaging, mice were treated with FAP-IL2v or vehicle, and treatment was repeated 3 days later. Subsequent PET/CT imaging was performed 3, 5, and 7 days after baseline imaging. In addition to in vivo PET imaging, ex vivo autoradiography, histology, and immunofluorescence staining were performed on excised tumors. B16-FAP tumors were clearly detected with [68Ga]Ga-DOTA-Siglec-9 PET/CT during the follow-up period, without differences in tumor volume between FAP-IL2v-treated and vehicle-treated groups. Tumor-to-muscle uptake of [68Ga]Ga-DOTA-Siglec-9 was significantly higher in the FAP-IL2v-treated group than in the vehicle-treated group 7 days after baseline imaging, and this was confirmed by tumor autoradiography analysis. FAP-IL2v treatment did not affect VAP-1 expression on the tumor vasculature. However, FAP-IL2v treatment increased the number of CD8+ T cells and natural killer cells in tumors. The present study showed that [68Ga]Ga-DOTA-Siglec-9 can detect B16-FAP tumors and allows monitoring of FAP-IL2v treatment.

Protein engineering and design in ion channels and receptors.

Acetylcholine receptors (AChRs) expressed at the neuromuscular junction synapses are typical allosteric proteins that shuttle between at least two stable conformational states: Closed (C) and Open (O). Agonist binding to their target sites on the receptor in the extracellular domain triggers a global C→O conformational change that results in an open channel pore that allows ion conduction. How the receptor senses the chemical signal of an agonist and communicates it to the channel pore, located ~50Šaway, are key to understanding the receptor function. AChRs are indispensable for muscle contraction and their neuronal homologues play critical roles in the nervous system function. In this chapter, using a combination of single channel patch-clamp, computational approaches, and genetic engineering, we elucidate the principles of design and engineering to quantify the fundamental thermodynamic parameters of AChRs that regulate ligand binding and channel opening. The receptor engineering principles outlined here for the neuromuscular AChRs are applicable to the broader class of ligand-gated ion channel proteins.

Simlukafusp alfa (FAP-IL2v) immunocytokine is a versatile combination partner for cancer immunotherapy.

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rßγ > IL-2 Rßγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rßγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.

Corrigendum: Combination of T-Cell Bispecific Antibodies With PD-L1 Checkpoint Inhibition Elicits Superior Anti-Tumor Activity.

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Robotic Roux en Y gastric bypass can be safe and cost-effective in a rural setting: clinical outcomes from a community hospital bariatric program.

Robotic Roux en Y gastric bypass (R-RYGB) is becoming more common due to the shifting trend toward robotic gastrointestinal surgery. The goal of this study is to determine if R-RYGB can be safely implemented at a robotic bariatric surgery program in a community hospital with similar results to laparoscopic RYGB (L-RYGB) in a cost-effective manner. A total of 50 R-RYGB procedures were performed with the Xi and the X da Vinci systems and compared with 50 L-RYGB cases by a single surgeon from October 2018 to January 2020 at an acute-care community hospital in a rural setting with a high-volume MBSAQIP-accredited program. A retrospective chart review was conducted with IRB approval and statistical analysis of 30-day morbidity, mortality, re-interventions, and resolution of co-morbidities, with financial analysis of cost reduction. Both groups were similar in age, gender, ASA class, co-morbidities, and body mass index (BMI). There was no mortality or anastomotic leak. The 30-day morbidity for R-RYGB was 10.0% with a re-operation rate of 4.0%. There were no conversions to open, and the mean hospital length of stay was 2.22 ± 1.19 days. There were no statistically significant differences between R-RYGB and L-RYGB with respect to any measured outcome, including intraoperative time (121.94 vs. 113.52, respectively; p = 0.1495). However, when incidences and percentages were used, R-RYGB had improved performance for most of the outcomes measuring safety. There was an average cost reduction of $816.90 per case (total saving of $40,845.00 for 50 cases) in the R-RYGB by transitioning from a hybrid approach to a totally robotic approach. R-RYGB appears to be as safe as L-RYGB and can be performed in a rural community hospital while maintaining a low complication rate, achieving a high co-morbidity resolution rate, and saving costs with a totally robotic approach.

Post-randomization for controlling identification risk in releasing microdata from general surveys.

Before releasing survey data, statistical agencies usually perturb the original data to keep each survey unit's information confidential. One significant concern in releasing survey microdata is identity disclosure, which occurs when an intruder correctly identifies the records of a survey unit by matching the values of some key (or pseudo-identifying) variables. We examine a recently developed post-randomization method for a strict control of identification risks in releasing survey microdata. While that procedure well preserves the observed frequencies and hence statistical estimates in case of simple random sampling, we show that in general surveys, it may induce considerable bias in commonly used survey-weighted estimators. We propose a modified procedure that better preserves weighted estimates. The procedure is illustrated and empirically assessed with an application to a publicly available US Census Bureau data set.

Laparoscopic Duodenal Switch Versus Roux-en-Y Gastric Bypass at a High-Volume Community Hospital: a Retrospective Cohort Study from a Rural Setting.

BACKGROUND: The classic duodenal switch (DS) represents a minority of bariatric procedures due to its high complexity and potential for complications. METHODS: A retrospective chart review was conducted on 100 laparoscopic DS cases from 2014 to 2018 at an accredited program in a rural community hospital and compared to 100 laparoscopic Roux-en-Y gastric bypasses (RYGB). Primary outcomes were 30-day morbidity and mortality. Secondary outcomes included anastomotic leak and remission of type 2 diabetes. RESULTS: There were more demographic risk factors for DS. The 30-day morbidity was higher for DS compared to RYGB (31% vs 13%, respectively; p = 0.0037). There was one mortality for DS and none for RYGB. There were statistically significant longer intraoperative times, greater EBL, and greater decrease in BMI for DS. The DS had a lower incidence of anastomotic ulcers (4% vs 13%, respectively; p = 0.0289), with a higher incidence of subsequent surgery beyond 30 days (21% vs 8%, respectively; p = 0.0160). There were 3 anastomotic leaks for DS and none for RYGB, although not statistically significant (p = 0.2463). The DS was more likely to eradicate hypertension, but the RYGB was more likely to eradicate GERD. There were no statistically significant differences for type 2 diabetes remission (92.1% vs 89.5%, respectively; p = 0.7239). CONCLUSION: Laparoscopic DS offers greater weight loss and hypertension remission, with lower incidence of anastomotic ulcers, but at the expense of greater morbidity and need for subsequent surgery, with no significant differences in type 2 diabetes remission when compared to RYGB in a rural community hospital program.

Combination of T-Cell Bispecific Antibodies With PD-L1 Checkpoint Inhibition Elicits Superior Anti-Tumor Activity.

T-cell Bispecific Antibodies (TCBs) elicit anti-tumor responses by cross-linking T-cells to tumor cells and mediate polyclonal T-cell expansion that is independent of T-cell receptor specificity. TCBs thus offer great promise for patients who lack antigen-specific T-cells or have non-inflamed tumors, which are parameters known to limit the response of checkpoint inhibitors. The current study deepens the understanding of TCB mode of action and elaborates on one of the adaptive resistance mechanisms following its treatment in vivo in humanized mice and syngeneic pre-clinical tumor models. Single-agent TCB treatment reduced tumor growth compared with controls and led to a 2-10-fold increase in tumor-infiltrating T-cells, regardless of the baseline tumor immune cell infiltration. TCB treatment strongly induced the secretion of CXCL10 and increased the frequency of intra-tumor CXCR3+ T-cells pointing to the potential role of the CXCL10-CXCR3 pathway as one of the mechanisms for T-cell recruitment to tumors upon TCB treatment. Tumor-infiltrating T-cells displayed a highly activated and proliferating phenotype, resulting in the generation of a highly inflamed tumor microenvironment. A molecular signature of TCB treatment was determined (CD8, PD-1, MIP-a, CXCL10, CXCL13) to identify parameters that most robustly characterize TCB activity. Parallel to T-cell activation, TCB treatment also led to a clear upregulation of PD-1 on T-cells and PD-L1 on tumor cells and T-cells. Combining TCB treatment with anti-PD-L1 blocking antibody improved anti-tumor efficacy compared to either agent given as monotherapy, increasing the frequency of intra-tumoral T-cells. Together, the data of the current study expand our knowledge of the molecular and cellular features associated with TCB activity and provide evidence that the PD-1/PD-L1 axis is one of the adaptive resistance mechanisms associated with TCB activity. This mechanism can be managed by the combination of TCB with anti-PD-L1 blocking antibody translating into more efficacious anti-tumor activity and prolonged control of the tumor outgrowth. The elucidation of additional resistance mechanisms beyond the PD-1/PD-L1 axis will constitute an important milestone for our understanding of factors determining tumor escape and deepening of TCB anti-tumor responses in both solid tumors and hematological disorders.

The PET-Tracer 89Zr-Df-IAB22M2C Enables Monitoring of Intratumoral CD8 T-cell Infiltrates in Tumor-Bearing Humanized Mice after T-cell Bispecific Antibody Treatment.

CD8-expressing T cells are the main effector cells in cancer immunotherapy. Treatment-induced changes in intratumoral CD8+ T cells may represent a biomarker to identify patients responding to cancer immunotherapy. Here, we have used a 89Zr-radiolabeled human CD8-specific minibody (89Zr-Df-IAB22M2C) to monitor CD8+ T-cell tumor infiltrates by PET. The ability of this tracer to quantify CD8+ T-cell tumor infiltrates was evaluated in preclinical studies following single-agent treatment with FOLR1-T-cell bispecific (TCB) antibody and combination therapy of CEA-TCB (RG7802) and CEA-targeted 4-1BB agonist CEA-4-1BBL. In vitro cytotoxicity assays with peripheral blood mononuclear cells and CEA-expressing MKN-45 gastric or FOLR1-expressing HeLa cervical cancer cells confirmed noninterference of the anti-CD8-PET-tracer with the mode of action of CEA-TCB/CEA-4-1BBL and FOLR1-TCB at relevant doses. In vivo, the extent of tumor regression induced by combination treatment with CEA-TCB/CEA-4-1BBL in MKN-45 tumor-bearing humanized mice correlated with intratumoral CD8+ T-cell infiltration. This was detectable by 89Zr-IAB22M2C-PET and γ-counting. Similarly, single-agent treatment with FOLR1-TCB induced strong CD8+ T-cell infiltration in HeLa tumors, where 89Zr-Df-IAB22M2C again was able to detect CD8 tumor infiltrates. CD8-IHC confirmed the PET imaging results. Taken together, the anti-CD8-minibody 89Zr-Df-IAB22M2C revealed a high sensitivity for the detection of intratumoral CD8+ T-cell infiltrates upon either single or combination treatment with TCB antibody-based fusion proteins. These results provide further evidence that the anti-CD8 tracer, which is currently in clinical phase II, is a promising monitoring tool for intratumoral CD8+ T cells in patients treated with cancer immunotherapy. SIGNIFICANCE: Monitoring the pharmacodynamic activity of cancer immunotherapy with novel molecular imaging tools such as 89Zr-Df-IAB22M2C for PET imaging is of prime importance to identify patients responding early to cancer immunotherapy.

iPSC model of CHRFAM7A effect on α7 nicotinic acetylcholine receptor function in the human context.

The α7 nicotinic acetylcholine receptor (α7nAChR) has been a promising target for diseases affecting cognition and higher cortical functions; however, the effect observed in animal models failed to translate into human clinical trials identifying a translational gap. CHRFAM7A is a human-specific fusion gene with properties that enable incorporation into the α7nAChR and, being human specific, CHRFAM7A effect was not accounted for in preclinical studies. We hypothesized that CHRFAM7A may account for this translational gap and understanding its function may offer novel insights when exploring α7nAChR as a drug target. CHRFAM7A is present in different copy number variations (CNV) in the human genome with high frequency. To study the functional consequences of the presence of the CHRFAM7A, two induced pluripotent stem cell (iPSC) lines (0 copy and 1 copy direct) were developed. The 0 copy line was rescued with CHRFAM7A transfection to control for genetic heterogeneity. As readouts for genotype-phenotype correlation, α7nAChR synaptic transmission and amyloid beta 1-42 (Aß1-42) uptake were tested. Synaptic transmission in the presence of CHRFAM7A demonstrated that PNU-modulated desensitization of α7nAChR currents increased as a function of CHRFAM7A dosage. CHRFAM7A mitigated the dose response of Aß1-42 uptake suggesting a protective effect beyond physiological concentrations. Furthermore, in the presence of CHRFAM7A Aß1-42 uptake activated neuronal interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) without activating the canonical inflammasome pathway. Lead optimization may identify more potent molecules when the screen has a model harboring CHRFAM7A. Incorporating pharmacogenetics into clinical trials may enhance signals in efficacy measures.