Despite the benefits of social support on the well-being of Black men across the life course, scholars are more closely examining the potentially negative outcomes associated with some social networks. As one social support system, the Black church frequently serves middle and old age Black men who identify as religiously involved. Yet, higher levels of religious involvement have also been associated with more church-related negative interactions. The present study utilizes a grounded theory approach to examine the negative interactions of religious middle and old age Black men. A semi-structured interview protocol is used to gather data from 35 Black men between the ages of 45 and 76. Analyses reveal that church-related negative interactions broadly fall within the following themes: (1) Ageism Within Intergenerational Churches, (2) People are Messy, and (3) Issues with Leadership. Since negative interactions can be more detrimental than social support is beneficial, health-related implications are discussed.


ABSTRACT IMPACT: A one-stage Bayesian multilevel model for meta-analysis integrating different survival data is introduced to complete the information synthesis without assuming proportional hazard. OBJECTIVES/GOALS: To develop a general modeling approach to perform efficient and robust meta-analyses using aggregated data (AD) for survival type endpoint and apply to a meta-analysis to examine the association between measurable residual disease (MRD) and disease-free survival (DFS) and overall survival (OS) in patients with acute myeloid leukemia (AML). METHODS/STUDY POPULATION: A Bayesian semi-parametric hierarchical model with a time-varying HR effect was presented. Three common types of survival information, including reconstructed survival data, the hazard ratio (HR) estimates with corresponding CIs and survival rates at specific time points, are synthesized such that all literature from the systematic review can contribute properly to the estimation and uncertainty quantification of the model parameters. The time-varying effects was modeled by assuming piecewise hazard risk and piecewise constant hazard ratio. The heterogeneity across studies was expressed by an additive random study effect and a random treatment-by-study interaction. The method was applied to a systematic review of 81 publications reporting on 11,151 AML patients. RESULTS/ANTICIPATED RESULTS: In simulation studies that the proportional hazard assumption is either valid or violated, the proposed method was efficient to achieve comparable performance to IPD meta-analysis, a gold standard approach, in estimating the survival rates, the restricted mean survival time at specific time points and median survival time with the point estimates close to the true values. When HR is not proportional over time, the proposed method was robust in estimating HR and significantly outperformed the classical random-effects meta-analysis. In the application to AML study, the average HR for achieving MRD negativity was 0.36  for OS and 0.37  for DFS. The association of MRD negativity with OS and DFS was significant and consistent across all subgroups. DISCUSSION/SIGNIFICANCE OF FINDINGS: The proposed novel method provided a flexible framework for meta-analysis of survival data, to accommodate various types of survival data in one model without assuming proportional hazards assumption. The findings of AML meta-analysis suggest that achievement of MRD negativity is associated with superior DFS and OS in patients with AML.


Staphylococcus aureus (MSSA). The eustachian valve is an embryological remnant of the inferior vena cava that has the potential to harbor a nidus leading to infective endocarditis. Eustachian valve endocarditis has been documented in the literature on less than 50 occasions and has yet to be documented in the presence of concomitant vertebral osteomyelitis. In this case, we present a 43-year-old male presenting with vertebral osteomyelitis caused by methicillin-resistant Staphylococcus aureus (MRSA). Persistent bacteremia prompted the identification of vegetative growth on a eustachian valve remnant. This case helps mend the gap in the literature by documenting the treatment considerations in a patient with eustachian valve endocarditis in the presence of osteomyelitis caused by MRSA.Infective endocarditis and vertebral osteomyelitis are rare infections, most commonly caused by methicillin-sensitive  Staphylococcus aureus (S. aureus) is the most common pathogen associated with both osteomyelitis and IE, with a proclivity of infections resulting from methicillin-sensitive strains [Infective endocarditis (IE) and osteomyelitis are rare and potentially life-threatening infections.  strains -3. Conco strains . In IE,  strains .S. aureus [Instances of embryological heart remnants contributing to endocarditis have been infrequently documented in the literature. Of those cases, less than 50 involve an embryological remnant of the inferior vena cava (IVC), known as the eustachian valve (EV). Persistent EVs occur in approximately four percent of the general population and are generally a benign and incidental finding [. aureus . To our A 43-year-old male presented to the emergency department via ambulance with altered mental status, visual hallucinations, and excruciating back pain as reported by an accompanying caretaker. On arrival, the patient was afebrile, mildly tachycardic (HR: 98 BPM), and borderline hypotensive (BP: 98/63 mmHg). Further examination revealed an ill-appearing, obese male in no acute distress. The patient was disoriented to place and time and was unable to follow simple commands. A detailed neurological examination could not be performed due to a lack of cooperation. The patient's vision was grossly intact and cardiovascular examination revealed a normal S1 and S2 with no murmurs, rubs or gallops. The patient had a history of poorly controlled insulin-dependent diabetes mellitus, end-stage renal disease on maintenance hemodialysis via right internal jugular vein (IJV) vascular catheter, persistent atrial fibrillation anticoagulated with apixaban, and bilateral below-the-knee amputation secondary to diabetic complications with chronic osteomyelitis from MRSA. The patient had a history of medical noncompliance and reported discontinuous medical care managed at multiple distant care facilities.A non-contrast computed tomography (CT) scan of the neuraxis revealed a destructive process in the thoracic spine at T7-T8 suspicious for discitis and osteomyelitis Figure  with unrS. aureus (MRSA). A CT-guided biopsy of the spinal abscess was scheduled but was later cancelled per the patient's request. The patient was managed on an in-patient basis for a total of 16 days with serial blood cultures. Repeat blood cultures at the end of week 1 remained positive for MRSA. The patient’s right IJV vascular catheter was removed and tested positive for MRSA. The catheter was replaced on two separate occasions and tested positive for MRSA both times. Vancomycin therapy was superseded by IV daptomycin  and rifampin (300 mg twice daily) due to persistent bacteremia.Magnetic resonance imaging (MRI) of the thoracic spine confirmed osteomyelitis at T7-T8 and a similar yet earlier infectious process occurring at T10-T11 Figure . Blood cBlood cultures remained positive for MRSA at the beginning of week 2. Concerns surrounding the persistent bacteremia prompted a transthoracic echocardiogram (TTE), which revealed the presence of a mobile vegetation on an embryological remnant of the IVC, an EV remnant, within the inferior aspect of the right atrium Figure . In-patiIE is a focal infection of the endocardium that has a high potential for systemic dissemination. In the United States, IE is rare and occurs at a rate of 11.4 per 100,000 adults ,8. CommoThe presence of structural heart defects and bacteremia should raise the index of suspicion for IE, but not all defects are outwardly evident. In this case, the patient did not display overt signs of structural heart defects, such as an audible murmur or patient-reported diagnosis. A broad screening effort included the use of a TTE to rule out the heart as a source of persistent infection. While TTE is less specific for right atrial pathology than transesophageal echocardiography (TEE) due to limitations of probe positioning, TTE remains the preferred initial screening modality ,13,14. FIn the setting of IE, bacterial dissemination to the spine most commonly occurs in the lumbar vertebrae . VertebrClinical treatment of coexisting IE and vertebral osteomyelitis relies on pathogen-directed therapy. In this case, the presence of MRSA in serial blood cultures limited effective therapy to narrow-spectrum IV antibiotics, such as vancomycin and daptomycin. A minimum of six weeks of therapy is recommended, with eight to 12 weeks warranted in treatment-resistant strains . NeverthA version of this article was posted on a preprint server (Authorea) on May 6, 2020 [Vertebral osteomyelitis in the presence of persistent or recurrent bacteremia warrants screening for active IE. This case report documents an embryological remnant of the IVC, known as the EV, as a source of IE in the setting of vertebral osteomyelitis. While this constellation of comorbidities is rare, this case underlines the importance of a thorough evaluation and persistent monitoring in the treatment of such persistent infectious conditions.


Dear Editor,For kidney transplant being the ideal solution for patients with end-stage kidney disease, there is an increasing demand for donated organs available for transplantation. According to the recent United Network for Organ Sharing (UNOS) update, there are more than 110,000 candidates on the kidney transplant waitlist, with the number growing every year [Our modern world is facing extraordinary circumstances while passing through a serious pandemic caused by the novel coronavirus (COVID-19) which may lead to multi-organ system failure & death . 198,589We retrospectively reviewed all renal transplant recipients registered in the UNOS database who had their transplants during the 1st wave of COVID-19 pandemic between 1st of March 2020 and 1st of December 2020. Patients who received kidney transplants from a deceased donor with a positive COVID-19 polymerase chain reaction (PCR) test within three months prior to transplant were included in our study.The STARFILES used in our study were: KIDPAN, KIDPAN_IMMUNE_DISCHARGE, and DECEASED_DONOR_DATA. Patients were followed up till the 4th of December 2020. Data about recipient factors , transplant factors  and donor factors  were collected. Outcome measured were patient and graft survival till the end of the follow-up.We found five renal transplant patients received kidneys from three deceased donors who tested positive for COVID-19 infection using PCR test. Details for baseline characteristics for transplant recipients and donors are shown in The results of our study highlight the importance of considering deceased patients secondary to COVID-19 infection as potential kidney donors. Many uncertainties surround kidney donation during the COVID-19 era. The guidelines’ recommendations about kidney donation show disparities, low-quality evidence and are difficult to implement in clinical practice. The American Society of Transplant Surgeons recommends suspending all living donations unless necessary. The deceased donation can be resumed if it is in the interest of the recipient . NHSB’s Literature rejected the possibility of transmission of COVID-19 through blood and blood products of vertical transmission to the fetus from COVID-19 infected mothers . Kwon etUtilizing kidneys from donors with infectious risks has the potential to become the standard of care in many centers . The urgOur report has certain limitations. Being retrospective in nature with the small number of patients included limits the ability to produce any causal interpretation. Another limitation is the short follow-up period. However, rejection episodes occur most often in the first six months after transplant. More studies are needed to address the short and long-term complications and outcomes for accepting kidneys from individuals who tested positive for the novel coronavirus. Additional work is urgently needed to clarify the post-donation health hazards among those patients and assure the safety of the patients. In conclusion, receiving kidneys from selected deceased donors who tested positive for COVID-19 infection may be safe and enable tapping in to a source of organ donation so far not considered.


Accelerated nodulosis (ARN) is a rare variant of rheumatoid nodules (RNs) that is characterized by a rapid onset or the worsening of RNs. It generally develops at the fingers in patients with rheumatoid arthritis (RA) receiving methotrexate (MTX). Few case reports have described ARN at an extracutaneous location.An elderly patient with long-standing RA was admitted to our hospital with acute respiratory failure. Computed tomography upon admission showed diffuse ground-glass opacities superimposed with subpleural reticular shadowing and honeycombing and multiple nodules in the lungs and liver. Despite the discontinuation of MTX and introduction of an immunosuppressive regimen with pulse methylprednisolone followed by a tapering dose of prednisolone and intravenous cyclophosphamide, the patient died due to the acute exacerbation (AE) of RA-related interstitial lung disease (ILD) following the parallel waxing and waning of a diffuse interstitial shadow and pulmonary and liver nodules. At autopsy, RNs were scattered throughout both lung fields in addition to extensive interstitial changes. RNs were also detected in the liver and kidneys. The foci of cryptococcosis were mainly identified in alveolar spaces. Based on the clinical and pathological findings, these nodules were most consistent with ARN because of acute increases in the size and number of previously detected pulmonary nodules.The present case is noteworthy because ARN was concurrently detected in multiple internal organs and may be associated with the AE of RA-related ILD. Classic rheumatoid nodules (RNs) occur in approximately 20 to 25% of patients with seropositive rheumatoid arthritis (RA). RNs develop as a later manifestation of active arthritic disease and are mostly identified in areas prone to mild repetitive traumatism, such as bony prominences, extensor surfaces, or regions adjacent to joints. Therefore, RNs commonly occur on the elbows and fingers, but rarely in the lungs or other internal sites. Risk factors for RNs are being male, smoking, ex-smokers, and elevated levels of serum rheumatoid factor (RF) and the anti-cyclic citrullinated peptide (anti-CCP) antibody , 2.Accelerated nodulosis (ARN) is characterized by the rapid onset or worsening of RNs and appears to be a variant of RNs. It was originally recognized as a complication of methotrexate (MTX) therapy in patients with RA because lesions often regressed after the cessation of MTX and recurred after a re-challenge . HoweverWe herein introduce a case of the simultaneous occurrence of ARN in the lungs, liver, and kidneys in addition to the AE of the usual interstitial pneumonia (UIP) subtype of RA-related ILD in a 74-year-old male with long-standing, anti-CCP antibody-positive RA.A 74-year-old male was transferred to our hospital with acute respiratory failure. He had been diagnosed with RA at the age of 57 years and had been treated with 12 mg per week of MTX, 1000 mg of sulfasalazine, and 100 mg of bucillamine at another hospital. RA disease activity had been maintained at a low level according to composite indices: the most recent simplified disease activity index and clinical disease activity index were 9.28 and 5.4, respectively. Previous radiography of the hands approximately 13 months before admission showed ulnar deviation of the left fingers as well as joint-space narrowing of the radiocarpal joint of the right hand  is infection [Aspergillus empyema [A blood culture obtained on the day of death yielded nfection . The inf empyema . TherefoThere were concerns that necrotizing granulomas were due to cryptococcosis rather than ARN. A previous case report described pulmonary cryptococcosis mimicking RNs in a patient with RA . SimilarIn summary, we herein present a male patient with long-standing RA who developed ARN in multiple internal organs. We speculate that the progression of ARN may have been associated with the AE of RA-ILD. Since there have not yet been any similar case reports, the present case will broaden the differential diagnoses of multiple pulmonary nodules, particularly nodules that show waxing and waning during the clinical course.


The FFCS platform has recently been developed by the Swiss Light Source macromolecular group. It is an integrated next-generation X-ray crystallography fragment-based drug-discovery pipeline for crystal soaking, handling and data collection. Over the last two decades, fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds. X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods. However, it is also often used with great success as a primary screening technique. In recent years, technological advances at macromolecular crystallography beamlines in terms of instrumentation, beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography. Here, the development of the Fast Fragment and Compound Screening (FFCS) platform, an integrated next-generation pipeline for crystal soaking, handling and data collection which allows crystallography-based screening of protein crystals against hundreds of fragments and compounds, at the Swiss Light Source is reported. Fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify promising chemical scaffolds for the development of lead compounds  but with high ligand efficiency. In order to progress towards high-affinity compounds, the binding affinity of hits can be increased by coupling fragments (‘linking and merging’) or derivatizing a single fragment (‘growing’). This process affords a greater understanding and control of the binding mode and pharmacokinetic properties of the resulting lead compounds compared with the development of HTS hits. To date, drug candidates arising from FBDD programmes have achieved considerable success, with six drugs approved by the United States Food and Drug Administration (USFDA) and more than 15 candidates currently in clinical trials .In contrast to HTS, FBDD utilizes small libraries (1000–3000 members) of simple molecules  to increase solubility. Commonly used techniques for fragment screening include surface plasmon resonance, nuclear magnetic resonance, thermal denaturation midpoint-shift assays and mass spectrometry. When evaluating the development potential of fragment hits, other lower-throughput techniques such as isothermal titration calorimetry, microscale thermophoresis, biochemical assays and X-ray crystallography can be used to provide information about binding affinity and thermodynamics, as well as binding location and orientation.et al., 1997et al., 2007et al., 2006et al., 2017et al., 2021et al., 2017et al., 2021et al., 2021et al., 2012et al., 2020In addition to its use in validating and characterizing the binding of fragment hits identified by other techniques, X-ray crystallography has achieved great success as a primary screening method. It is one of the most sensitive and reliable techniques for the detection of binding, and gives key structural information on fragment poise. In addition, X-ray crystallographic FBDD (xFBDD) can be ‘site-agnostic’, identifying and locating fragments bound both at known active sites and at allosteric sites. Thus, it allows druggability to be assessed for the whole accessible protein surface, without assumptions, and in the absence of known high-affinity binders. The use of X-ray crystallography for primary screening in FBDD was first demonstrated in 1997 (Verlinde The Swiss Light Source (SLS) macromolecular crystallography (MX) group has recently developed their own xFBDD platform, called the Fast Fragment and Compound Screening (FFCS) platform. In this publication, we present this newly established FFCS pipeline, focusing on hardware and software solutions.2.(i) Crystal growth: crystals are grown in SWISSCI MRC-3 plates, which are stored and imaged in a Rock Imager (Formulatrix) at 4°C or room temperature (RT).et al., 2017(ii) Crystal soaking: performed with an Echo 550  Crystal harvesting: assisted by a Crystal Shifter robot  Data collection: at one of the three SLS MX beamlines.(v) Initial data processing and structure solution: within the beamline computing environment.The FFCS pipeline was developed within the MX group at the SLS. The main aim of the FFCS project was the development of an integrated next-generation pipeline for crystal soaking, handling, and data collection and analysis, which would aid high-throughput drug-discovery processes in the pharmaceutical industry and academic laboratories. The FFCS pipeline includes the following steps Fig. 1.(i) CrysFFCS software suite, which allows easy experiment design, efficient bookkeeping and links all of the steps of the FFCS pipeline. Close collaboration with the group of Günter Fritz, University of Hohenheim, enabled the extension of the initial data processing and structure solution with an advanced data-evaluation pipeline developed in his group .The process, which involves handling hundreds of crystals and fragments/compounds, requires the creation of tools to make it faster, more efficient and more automated. The crucial components of the FFCS pipeline are the hardware setup and the dedicated in-house-developed software, the 3.In order to accommodate new equipment, the SLS Macromolecular Crystallization Facility (CF), which is localized in close proximity to the MX beamlines, was rearranged substantially. A separate space dedicated solely to the FFCS equipment was created and adapted accordingly. The FFCS hardware setup was to a large extent inspired by the XChem Fragment Screening facility at DLS, which is the largest facility for high-throughput crystallographic fragment screening that is tailored for academic users.et al., 2003The Crystallization Facility is equipped for robotic crystallization screening using sitting-drop vapour diffusion (or lipidic cubic phase) with a Mosquito robot (SPT Labtech), which is available at both 4°C and RT. Crystallization plates are barcoded, stored and imaged in dedicated 4°C or RT Rock Imager (Formulatrix) plate hotels. The Echo 550 noncontact liquid handler (Labcyte) is mounted on a specialized vibration-free table to prevent any interference during acoustic droplet injection experiments. To ensure longevity and to maintain high quality, FFCS fragment and compound libraries are stored in barcoded Echo Qualified 384-well or 1536-well COC plates (Labcyte) in a dedicated storage system. The StoragePod system (Roylan Developments) consists of storage pods housing the fragment libraries and a controller unit to purge the storage pods with dry nitrogen gas. This enables the storage of fragment and compound libraries in an inert, moisture-free and oxygen-free environment, preventing their dilution, precipitation and damage. The library storage plates are stored at room temperature, since repeated freeze–thaw cycles of DMSO-dissolved substances are associated with compound degradation  equipped with two dedicated video cameras which enable scanning of both the puck and, optionally, the pin barcodes.The harvesting of fragment/compound-soaked crystals is assisted by a Crystal Shifter (CS) robot (Oxford Lab Technologies). The CS device is a motorized An extended effort was put into the FFCS laboratory design to ensure the ergonomic use of space and to guarantee an optimal workflow. Devices are located in close proximity in an arrangement reflecting the steps in the experimental procedure. Solutions such as a liquid-handler vibration-free table and a dedicated library storage system bestow robustness and reliability to the pipeline and improve upon the already existing facilities.4.FFCS software suite facilitates experiment design and seamless step-by-step progression within the FFCS pipeline while efficiently record-keeping on behalf of the user. The FFCS suite consists of the FFCS database (FFCS DB) and four graphical interfaces: the FFCS GUI, Xi GUI, soakedMe GUI and shiftMe GUI  and picks crystals for soaking in the Xi GUI; these are accessible on the user console . The FFCS GUI sends input files for Echo and Crystal Shifter experiments, while the soakedMe (Echo PC) and shiftMe  GUIs report the results of soaking and fishing experiments back to the FFCS GUI.The in-house-developed UI Fig. 2. A dedic4.1.https://www.docker.com/). The FFCS DB benefits from a MongoDB (https://www.mongodb.com/) schema-less design, which does not enforce a strict format of metadata. Currently implemented FFCS DB collections are ‘Plates’, ‘Wells’ and ‘Libraries’. Each collection includes an implemented validator to check for required keywords for each insert operation. Moreover, operation is not permitted if one of the keywords does not exist or the keywords are of the wrong type. All Python software that needs access to data in the FFCS DB utilizes a dedicated client, which is tailored to retrieve specific data as required by the database.The FFCS DB system receives and stores all of the metadata produced by users throughout FFCS campaigns. The FFCS DB is deployed on a dedicated virtual machine (Linux OS) as a docker container (FFCS GUI is a frontend PyQt5 (Python 3.6) application developed to interact with documents stored in the FFCS DB. It can be accessed via a dedicated FFCS user console that utilizes the same group accounts  to authenticate users at the beamline consoles. This allows the storage of all FFCS project-related documents in the same working space as is used for X-ray diffraction data collection. The e-account FFCS folder includes (i) copies of the crystallization drop images, (ii) Echo and Crystal Shifter input files (in .csv format), (iii) a data collection spreadsheet compatible with the TELL GUI, user software which controls the TELL automatic sample changer , (v) a spreadsheet with FFCS experiment reports and (vi) fragment-library file(s) (in .sdf format).The FFCS GUI, the user is presented with a menu listing all ongoing FFCS campaigns associated with a given e-account (the campaign name functions as a unique identifier); alternatively, users can start a new campaign . The FFCS GUI tabs represent consecutive stages of a given campaign, namely ‘Plates’, ‘Cryo’, ‘Soaking’, ‘Redissolve’ and ‘Fishing’. Crystallization plates can be added to the ongoing FFCS campaign in the Plates tab. The plate ID can be typed in manually or it can be scanned with the barcode reader attached to the user console. The list of crystallization plates associated with the given FFCS campaign is retrieved by the Xi GUI and updated after the dispensing locations have been selected. The Cryo tab is an optional step of the FFCS pipeline and must be performed before the final soaking experiment. The Soaking tab allows the selected fragments list to be combined with the targeted crystallization drops . The ‘Export to Soak’ button triggers ZeroMQ (https://zeromq.org/) transfer of the soaking-definition file to the Echo PC and of the Fishing definition file to the Crystal Shifter PC. It also starts a soaking duration timer, which is automatically refreshed every minute in the ‘Soak Duration’ column. After soaking has been completed, the Echo experiment report is sent to the FFCS DB, the Soaking tab timer is stopped, and the soaking status displayed in the FFCS GUI is updated accordingly. The Redissolve tab is an optional step of the FFCS pipeline which was created on demand for a challenging project that required DMSO-free soaking  which allows the easy identification of drops with suitable crystals and the precise definition of the fragment-dispensing location for targeted soaking experiments. Upon startup the GUI (i) connects to the FFCS DB and identifies FFCS campaigns for a given user (based on the e-account name), (ii) extracts the barcodes of the crystallization plates assigned to a selected campaign and (iii) copies images from the Rock Maker storage to the dedicated FFCS campaign folder. Images of crystallization drops are displayed in the central Xi GUI window and the dispensing location can be assigned with a single mouse click  assist users in sending results back to the FFCS DB. The soakedMe GUI (Echo PC) sends an Echo dispensing status report, which includes error messages. The shiftMe GUI  combines results from Crystal Shifter fishing and the barcode reading station before sending the report back. Both soaking and fishing experiment results are displayed for user inspection in the FFCS GUI.We created a suite of ZeroMQ servers and clients and two dedicated GUIs to enable the easy transfer of files between computers within the FFCS pipeline. As mentioned in the previous section, upon user request the 5.DA+ data-acquisition (daq) software GUIs, namely the DA+ GUI  automation software package is available at all three MX beamlines . SDU is a software alternative to the user controlling the experiment with the DA+ GUI and is an integral part of the SLS MX distributed daq software stack, communicating with many other software instances such as the TELL GUI and the mxdb database. SDU data collection and processing options can be defined by users in a sample spreadsheet (the template can be downloaded from the SLS MX webpage); alternatively, beamline-specific default parameters are applied. The automatic data processing (adp) provides instantaneous feedback on a subset of collected data (the first ‘fast_xds’ step) as well as fully processed data for the total angular range. In the second ‘complete’ step users can select one of the following processing pipelines: the in-house-developed gopy, autoPROC  codes for the fragments from the FFCS DB and generates ligand coordinates and restraints using eLBOW from the Phenix suite , easing further downstream data analysis.The X-ray diffraction data are collected manually or automatically on one of the three SLS MX beamlines . Manual data collection is performed using the SLS MX 6.et al., 2011et al., 2016et al., 2016et al., 2020et al., in preparation). For both projects, the 500 most diverse fragments (selected based on the structure descriptor FragFP) and 50 project-specific fragments were selected from one of the FFCS pipeline fragment libraries . Once the soaking conditions had successfully been optimized, for each of the studied crystal systems two to three days of intermittent laboratory work were sufficient to perform soaking and crystal harvesting . The assisted harvesting with the Crystal Shifter robot resulted in an average of 50–60 crystals being fished per hour for both tested systems. These numbers may be higher for a trained user considering that both projects exhibited some skin formation on top of the crystallization droplets. The X-ray diffraction data sets were collected on beamlines PXI X06SA and PXIII X06DA either manually or with the SDU software. Both manual data collection and SDU resulted in 12–14 data sets with a full 360° rotation per hour. The resolution for diffraction data collected within the reported projects after optimization was 1.7 and 1.5 Å on average . Data sets processed with adp (with the in-house XDS-based gopy pipeline) were fed into dimmer workers which calculate Fo − Fc difference density maps and generate fragment coordinates. Subsequent steps of data analysis were performed using the data-evaluation pipeline developed in the group of Günter Fritz. In brief, the pipeline consists of the following steps: (i) the creation of chemical restraints for each fragment with AceDRG . An interactive helper script defines data source and destination paths and enables parallelization of the calculations. The entire fitting and evaluation method will be described and exemplified in detail elsewhere . The hit rates for the two example projects were very similar, at 6.2% and 6.4%, respectively, and showed well defined, tightly bound fragments  protein target . For crystal systems that require cryoprotection, additional rounds of testing are completed. Understanding the significance of the DMSO optimization step is necessary in order to perform a successful xFBDD campaign , resulting in 30 fragment-bound structures (Bedi et al., 2020The advantage of the flexible in-house-developed In recent years great progress has been made in beamline automation, data collection and automatic data processing. This has enabled the development of dedicated xFBDD screening pipelines, which are now offered by many synchrotron facilities. The high-throughput laboratory setup combined with easy-to-use software makes more advanced early drug-discovery experiments routine for both expert and inexperienced users. At the same time, synchrotron facilities have widely expanded their services well beyond standard X-ray data collection, providing pre- and post-experiment steps such as sample preparation and data analysis, all within the same synchrotron environment.Pesudomonas aeruginosa, 7qu3NqrF from PDB reference: 7qu5PDB reference: Klebsiella pneumoniae, 7qtyNqrF from PDB reference: 7qu0PDB reference:


Recent studies have also suggested a role for dam in protection against antibiotic stress, although this is not yet fully defined. We therefore evaluated the role of dam in the development of antibiotic resistance and triclosan-associated cross-resistance. DNA adenine methyltransferase (dam knockout compared to the parental strain. Known triclosan resistance-associated mutations in fabI were seen regardless of dam status, with an additional mutation in lrhA seen in the dam knockout. The expression of multiple antibiotic resistance-associated genes was significantly different between the parent and dam knockout post-resistance induction. Reversion rate assays showed that resistance mechanisms were stable. A significant impact on growth rate was seen in the dam knockout had a significant effect on growth, but its role in the development of antibiotic resistance is likely confined to those antibiotics using acrAD-containing efflux pumps. The whole-genome sequencing data of the strains used in this study are available from NCBI under the BioProject accession number PRJNA517874.dam)  and sulphonamides [dihydropteroate synthase (folP)]  . HoweverrhA in the dam knockout . This mutation causes an inframe insertion of leucine and aspartic acid. While the effect of the mutation is unknown, it is predicted to be deleterious by the Protein Variation Effect Analyzer (Provean) [LrhA belongs to the lysR family and contains a helix–turn–helix (HTH) DNA-binding domain (amino acids 11–68), which overlaps with the insertion mutation (between amino acids 10 and 11). Mutations in such domains in other HTH-containing DNA-binding proteins have been shown to decrease DNA-binding capability [lrhA is decreased as a consequence of this mutation it would no longer be able to repress rpoS to the same extent as the wild-type, which may contribute to the increased rpoS expression seen here. Within the TCS-resistant dam knockout a synonymous mutation was seen in pcnB. While deletion mutations of pcnB have been shown to confer resistance to high concentrations of chloramphenicol, ampicillin and kanamycin, the significance of this mutation, apart from a general contribution to altered fitness costs, is unknown [An interesting observation from this work is the identification of the insertion mutation in lknockout . This muProvean) . LrhA bepability . If the  unknown dam knockout strain compared to the parental strain prior to resistance induction, with MICs of 0.9 μg ml−1 v 0.4 μg ml−1, respectively. Post-TCS resistance, TCS-exposed knockouts and parent strains were able to grow at concentrations up to 100 times greater than the initial concentration. TCS acts by disrupting the synthesis of fatty acids by competitive inhibition of ENR. TCS interaction increases the affinity of ENR for nicotinamide adenine dinucleotide (NAD+), resulting in the development of the stable ternary complex ENR/–NAD/TCS. In this form, ENR is unable to synthesize fatty acids [dam knockout, the level of fabI is significantly increased .Marginally increased resistance to TCS was seen in the ty acids . In the ncreased . I, and d  [While n et al.  that damnolones) .


Near-Field Meta-Steering (NFMS) is a constantly evolving and progressively emerging novel antenna beam-steering technology that involves an elegant assembly of a base antenna and a pair of Phase-Gradient Metasurfaces (PGMs) placed in the near-field region of the antenna aperture. The upper PGM in an NFMS system receives an oblique incidence from the lower PGM at all times, a fact that is ignored in the traditional design process of upper metasurfaces. This work proposes an accurate optimization method for metasurfaces in NFMS systems to reduce signal leakage by suppressing the grating lobes and side lobes that are innate artifacts of beam-steering. We detail the design and optimization approach for both upper and lower metasurface. Compared to the conventionally optimized compact 2D steering system, the proposed system exhibits higher directivity and lower side-lobe and grating lobe levels within the entire scanning range. The broadside directivity is 1.4 dB higher, and the side-lobe level is 4 dB lower in comparison. The beam-steering patterns for the proposed 2D compact design are experimentally validated, and the measured and predicted results are in excellent concurrence. The versatile compatibility of truncated PGMs with a low gain antenna makes it a compelling technology for wireless backhaul mesh networks and future antenna hardware. Any error due to antenna misalignment should be avoided to establish a robust link quality in these networks. Thus, a medium/high-gain 2D beam-steering-enabled antenna system with a planar profile is desirable to ease the deployment and cope with the wind-induced movements in the lampposts and other 5G small cell installation sites. These antennas must have the ability to transmit and/or receive in any arbitrary direction within a large conical region.The growing hope and hype around the Internet of Things (IoT) and constantly increasing demand for ubiquitous coverage with ultra-high-speed data over mobile networks has created a new wave of technological revolution in 5G cellular communication. The mobile backhaul technology, primarily used to feed massive data to the end-users, connects the network hub to the base stations wirelessly. It is the most cost-effective and versatile solution to connect 5G base stations to the core network to enable flexible and easy installation of base stations in ad-hoc networks, supporting large crowd gatherings such as concerts and sports events. These backhaul networks depend on the line-of-sight communication between two fixed antenna terminals with point-to-point data transmission3. Due to the limitations associated with traditional beam-steering methods, several unconventional technologies have been investigated, including optical beamformers4, liquid crystal-based electronic beam-steering5, and metasurface antennas and lenses8. In a basic comparison, the passive beam-steering antennas (without using active RF components) have certain advantages over active beam-steering antennas. The former is relatively cheaper, has better linearity and scalability, low noise, high power handling capability, broader bandwidth, and low DC power consumption, making them suitable for emerging commercial applications.Conventional mainstream configurations for beam-steering include mechanically moving a fixed beam antenna on a gimbal and electronically steering beams of phased arrays. The mechanical steering solutions are bulky, and a more sophisticated system needs expensive motors to ensure the reliability of vital data links. Active electronically scanned arrays are the most agile front-end systems, but because of the high-cost active Radio-Frequency (RF) components, their use is limited to high-end applications11. The concept was first introduced and demonstrated in 201712 and since then has been extended by various research groups16. The configuration of the classical NFMS system is shown in Fig. Near-Field Meta-Steering is a state-of-the-art passive beam-steering antenna technology inspired from the optical-beam scanning principle that is implemented using Risley prisms17 and a pair of rotating PGMs placed above the base antenna in its near-field region. The first PGM tilts the antenna beam by introducing a phase shift in the antenna electric near field. The beam-tilt angle is controlled through the gradient of phase shift introduced by the PGM. Conventionally, the two PGMs in an NFMS system are identical and introduce the same phase gradient when placed on the antenna with planar phase fronts. In the NFMS systems, depending on its orientation, the second PGM can either increase or decrease the effective phase gradient and hence the antenna beam tilt angle. The base antenna remains fixed, and the two PGMs are co/counter-rotated to steer the antenna beam to any location within a wide 2D conical scanning range. The NFMS antenna technology holds several advantages over conventional steering methodologies. The base antenna being entirely stationary obviates the need for RF rotary joints. Since the entire phase shifting is carried out in the near-field region, the system is extremely low-profile, making it aesthetically pleasing and easier to be mounted on moving platforms. The PGMs can be rotated with a low-power stepper motor, and the total volume of the system is fixed at all times, even when the beam is steered.It consists of medium/high-gain base antennas that can create planar phase fronts on the aperture21. Conventionally, both upper and lower PGMs are designed using the same unit cell-based approach. However, the upper PGM receives an oblique incidence at all times, which calls for a different design and optimization strategy to achieve overall optimal 2D beam-steering performance.The periodic arrangement of physical cells, referred to as supercells, in PGMs generates periodic grating-type lobes. In addition to these grating lobes, the sidelobes appearing within antenna patterns when the beam is steered to larger elevation angles result in signal leakage, which is a concern for most wireless applications. To ensure efficient steering performance, the PGM-pair should be designed strategically. Besides, the sidelobes can be controlled by judiciously tailoring the arrangement of elements in the periodically repeating supercell of the PGMs. The lower metasurface atop the base antenna typically receives a normally incident wave and produces an output beam at an offset angle same as the gradient of the PGM. The design of such PGMs is straightforward and elaborately explained in literature22 has been used to optimize the lower PGM for normally incident plane waves. The upper PGM is optimized assuming obliquely incident plane wave with an angle of incidence same as the phase-gradient of the first PGM. Two compact beam-steering systems are modeled using a medium/low gain RCA as the base antenna. System-I has a pair of conventionally optimized PGMs . System-II has lower PGM optimized for normal incidence, and the upper PGM optimized for oblique incidence. The two systems are then compared based on their steering radiation performance. A prototype of the steering system that has better beam-steering performance is fabricated and measured. The results predicted by simulations in CST MWS are experimentally validated.In this work, we propose an innovative and more logical methodology to design and optimize PGMs in an NFMS system by considering the nature of the incident plane wave. The novelty of this work is to design upper PGM with cells having an excellent performance for the obliquely incident electric field. This is the first NFMS design that uses separately characterized cells for the upper and lower PGMs. A Floquet analysis-based Covariance Matrix Adaptation-Evolution Strategy (CMA-ES) optimization similar to the one presented in Ref.12. One of the modes, where beam is steered in both elevation and azimuth plane is achieved by fixing lower PGM in addition to the base antenna and only rotating the upper PGM. The optimization strategy proposed here, however, can also be implemented without any changes, for other steering modes where both metasurfaces are simultaneously rotated .The NFMS system can be operated in three steering modesThe PGMs are composed of periodic supercells designed using Phase-Transforming Cells (PTCs) arranged in a pre-defined sequence. The lower PGM is designed to tilt a normally incident plane wave at its input to a entclass1pt{minimaEach PTC comprises four metal layers (circular patches on top and bottom layer and square patches in the two middle layers) and three dielectric layers of Taconic TLY-5 . This concept is extended to optimize a smaller periodic supercell to enhance performance of a large metasurface, thus reducing computational cost and saving time and resources22.Floquet based optimization is a physics-oriented approach that acquires its concept from Floquet mode analysis of periodic structuresz-axis. For upper PGM, the supercell was excited with an obliquely incident TE(00) mode. The orientation of oblique incidence is defined in terms of elevation angle x-axis was generated using an array calculator in CST by implementing pattern multiplication to the far-field of a single supercell. An excellent correlation exists between the magnitudes and directions of the Floquet modes predicted by Floquet analysis of supercell and magnitudes and directions of the lobes in the far-field pattern obtained using the supercell simulation together with array calculation. Floquet space analysis of the two supercells reveals that only three out of five propagating modes in the transmission region are significant while the rest are evanescent. The knowledge derived from this analysis presents us with a possibility of controlling the side-lobes and grating lobes by optimizing the coupling of incident energy in the propagating modes22.We simulate the supercells with periodic boundary conditions and Floquet port excitation in CST MWS. For lower PGM, the supercell was excited with a broadside TE(00) mode propagating along the DM), and UDM). The value of The dimensions of the circular and square metal patches in both lower and upper supercells are optimized to bring 9 undesired modes (UDMs) below Since the optimization variables are continuous, the optimization algorithms inbuilt in CST work reasonably well. The initial dimensions from Table x-direction and 36 times along y-direction. The predicted radiation pattern for initial and optimized lower PGM is compared in Fig. The magnitude of grating lobes before and after optimization are also provided in Table Optimizing electrically large PGMs involves highly non-linear objective functions that exhibit an epistatic behavior due to strong mutual coupling and other propagation effects. Such design problems require a full-wave simulation-based derivative-free optimization approach. Evolutionary Algorithms (EAs) are a class of derivative-free optimization with superior exploration skills to search intractably large spaces. They can successfully achieve an optimal solution with a high probability for complex, multi-dimensional problems, operate with continuous, discrete as well as mixed parameters, are conducive to parallel computation, which significantly reduces the optimization run time by distributing the task among multiple computers. Their outcome is independent of the initial guess . Some of the widely used optimization methods in the field of Antenna and Propagation include Genetic Algorithm (GA), Particle Swarm Optimization (PSO), CMA-ES. GA and PSO optimization methods are susceptible to hyperparameters of the algorithms and require tedious parameter tuning, which is a challenging task. A better choice of the algorithm’s hyperparameters adapted to each function and dimension can seriously influence the final result. Since the CMA-ES method does not require tedious parameter tuning, the choice of the strategy to be adopted while setting the internal parameters is entirely automated. Therefore, it is more convenient than algorithms such as PSO and GA and hence is chosen in this work.26. RCAs are simple, efficient, and planar antennas with high directivity. They can be designed for both liner and circular polarizations. Low-profile medium-to-high gain RCAs find several applications in high-speed local wide-area networks and front-end antennas for point-to-point communication systems, such as commercial access and backhaul networks17. We uses a compact, strongly truncated RCA as a source of EM radiation that is known for its planar phase fronts since they have reasonably uniform phase in their aperture electric field. The base antenna is linearly polarized with the electric field parallel to the y-axis.We demonstrated the complete system by developing a compact NFMS system. The base antenna is a resonant cavity antennas (RCAs), which is also referred to as leaky-wave antenna, Fabry–Perot antenna and electromagnetic bandgap (EBG) antenna in the literatureA classical single layer RCA with a uniform dielectric superstrate composed of a circular disc of Rogers TMM 10 placed a half-wavelength , beyond which the beam goes in the lower invisible hemisphere until The configuration of the compact NFMS system with three-dimensional model is shown in Fig. We designed two compact beam-steering systems of same dimensions as specified in Fig. The elevation pattern cuts for System-I and System-II are compared in Fig. mentclasspt{minimaThe prototypes for the PGM pair of System-II along with the single-layer uniform-superstrate RCA were fabricated as shown in Fig. The fabricated components are assembled into compact near-field meta-steering system whose steering performance is measured for several configurations to validate numerically predicted results. The primary objective of the proposed design methodology is to demonstrate the steering of antenna beam in both azimuth and elevation planes, with reduced side-lobes and grating lobe levels and simultaneously maintain a fairly stable directivity.We first measure the performance of classical RCA prototype and compare with the simulated results as shown in Fig. The measured and simulated results are in good agreement. The antenna exhibits a reflection less than The first antenna assembly comprises of lower PGM and the RCA as shown in Fig. A photograph of the complete system is shown in Fig. The beam-steering antennas envisage a critical role in mobile backhaul technology. The electronically scanned antenna systems predominantly rely on active components, are complex and expensive. The NFMS antenna systems are an attractive alternative to traditional lossy beam-steering arrays with nearly equivalent performance with passive components. The absence of active elements such as phase shifters and amplifiers unlocks the potential for use in power-limited mobile environments. NFMS systems made of a base antenna and pair of PGMs use an approach to design both PGMs using cells that have excellent performance for normally incident field, which is not valid for the upper PGM. A Floquet analysis based CMA-ES optimization is implemented to first optimize the lower PGM for normal plane wave incidence and then to optimize upper PGM for oblique plane wave incidence to ensure minimal scattering in unwanted direction because of the coupling between different cells in the PGMs. A compact NFMS designed following the approach indicate ability to steer medium-gain beam to the maximum elevation angle of A low-cost compact beam-steering system is developed using a pair of truncated PGMs and a medium gain RCA. PGM-pair is optimized using Floquet based analysis and PSO algorithm. Analysis proved that an NFMS system has better steering performance when upper PGM is optimized for oblique incidence, and lower PGM is optimized for normal incidence compared to when both upper and lower PGMs are optimized for normal incidence. The planar profile of the designed steering system facilitates encapsulation into a cylindrical cavity. The metasurfaces are compact and lightweight and can be easily rotated with a pair of low-power stepper motors. Maximum directivity of the antenna system is 17.8 dBi, and it can be used to scan any elevation angle up to


By applying geneshot to two independent previously published cohorts, we identify microbial genomic islands consistently associated with response to immune checkpoint inhibitor (ICI)-based cancer treatment in culturable type strains. The identified genomic islands are within operons involved in type II secretion, TonB-dependent transport, and bacteriophage growth.Researchers must be able to generate experimentally testable hypotheses from sequencing-based observational microbiome experiments to discover the mechanisms underlying the influence of gut microbes on human health. We describe The online version contains supplementary material available at 10.1186/s13059-021-02355-6. Observational studies on the human gut microbiome using next-generation sequencing (whether 16S rRNA amplicons or whole genomic sequencing) have firmly established that the human gut microbiome affects health, disease, and response to treatments. The current analysis methods for whole-genome shotgun (or “WGS”) microbiome data have struggled to identify the underlying causal mechanisms, which are the critical next step to translate microbiome science into novel therapies. Identifying candidate mechanisms from observational studies is challenging because of the immense diversity of bacteria , widesprOne proposed solution is gene-level metagenomics: analysis with respect to abundance and presence of microbial protein-coding genes , but thigeneshot. geneshot is an end-to-end analysis workflow for microbiome experiments using CAGs as the fundamental unit of analysis. In brief, WGS data from each specimen is preprocessed to remove human reads and assembled de novo; predicted protein-coding genes are then deduplicated to make a reference gene catalog; the abundance of each gene in each specimen is estimated by the assignment of raw reads against that gene catalog after rigorous handling of multiply-mapping reads [We have incorporated the tactic of identifying and combining protein-coding microbial genes into CAGs into a computational analysis pipeline for gene-level metagenomic analysis called ng reads ; genes ageneshot to reanalyze data from metagenomic studies in people who have received immune checkpoint inhibitors for cancer. Immune checkpoint inhibitors (ICIs) are a class of immunotherapies that can induce robust and long-lasting protection from cancer [In this study, we employ m cancer –11. Howem cancer –11. Idenm cancer –14. Furtm cancer , 13, 15.geneshot to analyze the published datasets from microbiome studies investigating the stool microbiome of participants receiving ICI therapy for metastatic melanoma treatment [Odoribacter splanchnicus and Gemmiger formicilis (which were found to be more abundant in responders), as well as Coprococcus comes (which was more abundant in progressors)  that are suitable for targeted hypothesis testing .To provide genomic and functional context for these ICI-associated CAGs, we next aligned the dataset to 1886 reference genomes from the taxonomic families shown in Additional file geneshot is de novo assembled protein-coding gene sequences, which are dimension-reduced via co-abundance clustering. One advantage of this gene-level approach to metagenomic analysis is a reduced reliance on reference databases (which are often incomplete) or inaccurate ontological hierarchies . Moreover, by implementing an improved method for CAG construction, geneshot is able to analyze the gene-level abundances without being restricted to organismal groupings (such as MAGs or “metagenomic species”).In contrast to other end-to-end pipelines, the unit of analysis that underlies geneshot’s integration of reference and taxonomic databases is oriented around deriving maximum benefit from the sparsely sequenced and annotated uncultivated organisms within the gut. After identifying CAGs associated with an outcome, we then consider where the member genes of the associated CAGs have been observed in reference genomes. The actual organism representing these genes in a given specimen need not have a reference genome, just a somewhat related genomic island. Thus, geneshot maximizes what can be inferred from reference databases without being dependent upon the same references for the identification of relevant genomic islands.geneshot can be used widely to enhance the quality of microbiome research, while also providing insights which may contribute to the development of microbiome-based therapeutics for cancer and other human diseases.Finally, by wrapping together the set of tasks required for comprehensive gene-level metagenomic analysis using the Nextflow workflow management system , we provgeneshot using a series of bioinformatic processes which are all coordinated into an overarching workflow using the free and open-source Nextflow [geneshot can be found in the GitHub repository https://github.com/Golob-Minot/geneshot under the open-source MIT License, with documentation provided in the associated wiki.The process of performing gene-level metagenomic analysis is implemented by Nextflow  workflowgeneshot is as follows  and alignment coverage (default 50%) to retain only the centroids from each cluster of coding sequences.The preprocessed WGS reads from step 1 are then aligned against the gene catalog from step 3 in order to estimate the relative abundance of the organisms in each specimen which encode each gene. The alignment is carried out with DIAMOND (in blastx mode). Those alignments are then processed by FAMLI in order to resolve any reads which align equally well to multiple references by picking a single reference using an expectation-maximization approach that uses a target metric of evenness of sequencing coverage across each reference as the target metric .The relative abundance of each gene from the catalog across each specimen is computed as the depth of sequencing across each gene  divided by the sum of sequencing depth across all genes for that specimen. This provides a relative abundance estimate for the genomes encoding each gene in the source specimen that is adjusted for gene length.Using the relative abundance of each gene across each specimen, genes are clustered using average linkage clustering and the cosine distance metric, applying a fixed distance metric to join genes into co-abundant gene groups (CAGs). Each gene from the catalog which is detected in at least one sample belongs to one and only one CAG. Two complementary approaches are used to provide a computationally tractable approach to this process: (a) genes are clustered in sub-groups (shards) which are iteratively combined over five sequential rounds and (b) an approximate nearest neighbor algorithm is used to rapidly index and retrieve co-abundant gene “neighborhoods” to limit the search space for expensive computation of pairwise distance matrices as described previously . To mitiIndependently from steps 4–6, the gene catalog may be optionally annotated by taxonomic classification using DIAMOND (which assigns the lowest common ancestor of top hits in blastp mode), as well as functional annotation using eggNOG-mapper. These annotation steps are optional and are only executed if the user provides the reference databases required by either tool.If the user provides a formula for statistical analysis using the variables defined in the manifest , then the number of reads mapped to each CAG will be modeled using a beta-binomial model with that formula describing the logit of the expected relative abundance of the CAG. The model is fit using corncob . If taxogeneshot can be found in the associated documentation (https://github.com/Golob-Minot/geneshot/wiki/Output-Files).In the final step, all of the outputs from each step are aggregated into HDF5 format, which organizes multiple tables into a single file using an internal organization structure to identify each of the elements of the results. The updated description of all data output by The overview of the analytical tasks performed by geneshot, users must prepare (1) a manifest in CSV format with columns for “specimen,” “R1,” “R2,” and any other additional metadata needed for statistical analysis and (2) the set of FASTQ files (gzip-compressed) listed in the manifest, with forward and reverse reads listed separately in the “R1” and “R2” columns, respectively. In this format, a single specimen may be made up of multiple FASTQ file pairs by listing those files across multiple rows.In order to run geneshot on the available computational resources, start by installing the workflow management tool Nextflow and configuring it. The complete documentation for Nextflow can be found at https://www.nextflow.io/docs/latest/getstarted.html. The process of configuring Nextflow is accomplished by saving a “nextflow.config” file in the user’s home directory which describes the compute resources which should be used , and which will be referenced when running geneshot.In order to run geneshot can be found on the wiki associated with the tool at https://github.com/Golob-Minot/geneshot/wiki. In order to run geneshot, the user starts with the command “nextflow run Golob-Minot/geneshot” and appends any of the flags shown in Additional file The complete set of instructions for running geneshot is performed using reference databases for DIAMOND and eggNOG-mapper. The databases used for this analysis were generated based on the documentation in the associated tools and are available on AWS S3 at the following locations:Taxonomic annotation is based on a DIAMOND index of NCBI’s RefSeq genome database generated on January 15, 2020: s3://fh-ctr-public-reference-data/tool_specific_data/geneshot/2020-01-15-geneshot/DB.refseq.tax.dmndFunctional annotation is based on eggNOG-mapper v5.0 database downloaded on July 17, 2020: s3://fh-ctr-public-reference-data/tool_specific_data/geneshot/2020-06-17-eggNOG-v5.0/eggnog_proteins.dmnd and s3://fh-ctr-public-reference-data/tool_specific_data/geneshot/2020-06-17-eggNOG-v5.0/eggnog.dbAnnotation of the gene catalog generated by geneshot are available publicly as Docker images which are automatically generated from publicly available Dockerfile source code. The images used by the current version of geneshot are listed in Additional file All of the component tools used by geneshot output against a set of reference genomes was executed the Annotation of Microbial Genomes by Microbiome Association (AMGMA) tool. This tool aligns genes from the geneshot gene catalog against a user-specified set of microbial genomes and then calculates the summary metrics for each genome including the proportion of the genome which is aligned by genes which are associated with a given parameter. The genome alignment figures in this manuscript show the position of the genome alignment for the set of genes contained in the CAG of interest, as well as the location and annotation of genes recorded in GFF format within the NCBI RefSeq database. Alignment is performed using DIAMOND (using the dependencies shown in Table S2) with the complete set of Python code used to aggregate alignment results available at https://github.com/FredHutch/AMGMA/. An example of alignment to de novo assembled contigs, performed with the same set of steps, is shown in Additional file The process of aligning genes from the n=25), PRJNA397906 [n=44), and PRJNA399742 [n=172). The public metadata for PRJEB22893 [geneshot results published alongside this paper.The data analyzed in this manuscript consists of the publicly available FASTQ records and associated metadata available for the BioProjects PRJEB22893  (n=25), NA397906  (n=44), NA399742  (n=172).JEB22893  did not Additional file 1: Figure S1. Summary of gene-level metagenomic analysis results generated by geneshot. The results for each specimen across all cohorts is shown in comparison to the number of sequence reads for each specimen (x-position) including the number of genes detected by de novo assembly of each individual specimen (A), the proportion of reads from each specimen which align uniquely to the de novo gene catalog (B), and the number of genes from the gene catalog detected by alignment (C). The distribution of genes across CAGs of different sizes is shown in (D), with the horizontal axis showing the CAG size range and the vertical axis showing the number of genes belonging to CAGs within that size range. (E) shows the relative abundance (log10) of the CAGs with the highest relative abundance  across all specimens , with hierarchical clustering by relative abundance across rows and columns. Figure S2. Visual depiction of CAG-level abundance calculation. Each CAG consists of a group of genes, each of which is detected in each specimen by alignment of WGS reads. The abundance of genes from each CAG are shown in (A) and (C) in units of the absolute number of reads aligned uniquely from each specimen. The ‘depth’ of sequencing of each gene is a commonly-used length-normalized measure in which the total number of bases from all aligned reads is divided by the length of each gene. Genes are grouped into CAGs based on average linkage clustering using the cosine distance metric computed from the vector of sequencing depth values across all specimens. The abundance of each CAG is computed as the sum of sequencing depth for all genes in the CAG, divided by the sum of sequencing depth of all genes detected. That proportional abundance metric is shown for each CAG in (B) and (D). Specimens are arranged in alphabetical order on the horizontal axis, with abundance measures for each specimen on the vertical axis. Figure S3. Volcano plot showing the distribution of association estimates for CAGs with ICI response. Each point represents a single CAG, showing all CAGs which contain at least five constituent genes. The horizontal axis shows the estimated coefficient of association with ICI response . The vertical axis shows the p-value for that coefficient of association for each CAG after adjustment for multiple hypothesis testing with the FDR-BH procedure. Figure S4. Taxonomic annotation of CAGs associated with ICI response. The taxonomic annotation of genes found in the 50 CAGs (columns) which were most consistently associated with ICI response (after applying a minimum size threshold of 30 genes), are shown in (A) with shading indicating the proportion of genes assigned to the indicated taxon (rows). The Wald statistic summarizing the association with ICI response is shown for taxa in (B) and for individual CAGs in (D). A taxonomic tree annotating those organisms is shown in (C), and CAG size is shown in (G). Figure S5. Visual depiction of the aggregation of CAG-level ICI-response association metrics by taxon. For each organism, the lower panel shows the estimated coefficient and -log10 p-value for the association of each CAG with ICI response. Every CAG either contains at least one gene which was taxonomically annotated to the indicated organism (orange), or there were no genes in that CAG with that taxonomic annotation (blue). The aggregate taxon-level ICI-response association for each organism is shown in the upper panel from each subplot, with the estimated coefficient +/- standard deviation indicated in the subplot title. The vertical grey line in each subplot marks the taxon-level estimated coefficient value. Figure S6. A putative bacteriophage of Faecalibacterium prausnitzii is associated with ICI non-response, in contrast to an associated bacteriophage defense island that is associated with ICI response. The genomic coordinates of ICI response-associated CAGs are shown for two strains of F. prausnitzii (as in Fig. Figure S7. Example of CAG alignment to de novo assembly. The alignment coordinates for the alignment of a single CAG against the contigs generated by de novo assembly from this dataset is shown alongside the eggNOG functional annotations for the underlying genes. All alignments covered the entirety of the gene catalog sequence at >99% amino acid identity. The 14kb physical region shown in this plot encompasses all such high quality alignments for this CAG in this specimen.Additional file 2: Table S1. In order to run geneshot, the user starts with the command “nextflow run Golob-Minot/geneshot” and appends any of the flags.Additional file 3: Table S2. The images used by the current version of geneshot.Additional file 4. Review history.


Two-dimensional metal-organic frame-works 2D MOFs) have been explored intensively in the fields of materials science, chemistry and nanotechnology over the past decade . Their uD MOFs haAlthough top-down methods  or bottom-up methods  have been widely investigated for the fabrication of MOF nanosheets , it is set al., ‘Ultrathin metal-organic framework nanoribbons’ [In a recent work published in NSR by Wang ribbons’ , the resThe as-prepared CoBDC NRBs were used for the application of DNA detection. With the single-stranded DNA (ssDNA) probes on their surface, the CoBDC NRB was successfully used as the sensing platform. The quantitative detection of the target DNA can be achieved by measuring the fluorescence intensity of the probe. For the fluorescence quenching testing, the results showed that the CoBDC NRBs exhibit better performance than the bulk CoBDC MOFs materials Fig. g. ComparOverall, this work represents very important progress in MOF chemistry and 2D nanoscale materials through the facile and straightforward approach to preparing ultrathin 2D MOF materials. This pioneering work will significantly facilitate the development and implementation of 2D multifunctional MOF materials for diverse applications that will be beyond those developed on gas separation, catalysis, sensing and membrane devices in the future.Conflict of interest statement. None declared.


Case Presentation. A female with protein C deficiency, a severe bleeding disorder characterized by thrombosis formation, was diagnosed with GHD at age four years. She has been receiving daily protein C infusion through a permanent port since the newborn period. GHD was treated with daily IV rhGH boluses given through the port following protein C infusion. She has reached a growth rate of 12 cm/year and had no side effects. Surprisingly, serum insulin-like growth factor-1 (IGF1) levels did not rise despite an excellent clinical response.  The standard of treatment for pediatric growth hormone deficiency (GHD) is daily subcutaneous recombinant human growth hormone (rhGH) injections. The efficacy of rhGH treatment given as daily intravenous (IV) boluses is not known.  IV administration may be an alternative route for GHD treatment in eligible patients with permanent vascular access. A rise in serum IGF1 levels may not be needed to achieve the growth-promoting effect of rhGH. Short stature is one of the most common referral reasons to pediatric endocrinology clinics. Among the various etiologies of short stature, growth hormone (GH) deficiency is the most common endocrine cause. Growth hormone deficiency (GHD) is estimated to have a prevalence of 1 in 4,000 to 10,000 and can be due to genetic defects, acquired causes, or idiopathic. Untreated GHD may have a profound impact on the overall health of the affected children with lasting consequences beyond childhood. Presenting symptoms and signs vary depending on the age of onset, but children with GHD usually come to medical attention with decelerated or stunted linear growth, delayed bone development, and delayed puberty. Screening tests for suspected GHD include measurement of serum growth factors, particularly insulin-like growth factor (IGF)-1, which is also used to assess adherence and monitor safety and as a guide to titrate GH dose during treatment. IGF1 is the primary mediator of GH-stimulated bone growth. Currently, the standard treatment of GHD involves daily subcutaneous injections of recombinant human growth hormone (rhGH). However, this administration route may be a source of stress in parents of children with severe underlying bleeding pathologies because of cutaneous bleeding risk. Although the likelihood of hitting a large vein in subcutaneous fat is practically zero, the parental anxiety associated with this may lead to nonadherence to GH treatment in such patients. There is a lack of knowledge regarding the efficacy and safety of daily intravenous (IV) administration of rhGH to treat GHD. We report the case of a patient with protein C deficiency who was diagnosed with GHD and successfully treated via daily IV rhGH boluses through an established port. To our knowledge, the IV route has not been previously used for long-term rhGH replacement therapy.A 2-year 5-month-old female was referred to for evaluation of short stature. She was born at term with a birth weight of 3845 grams and a length of 51 cm. Her history was notable for prolonged Neonatal Intensive-Care Unit (NICU) stay due to disseminated intravascular coagulation (DIC) characterized by multiple skin bruises and respiratory distress. She required a chest tube and G-tube placement to treat chylothorax and feeding problems, respectively, and transfusion of multiple blood products to manage DIC. After an extensive workup, she was diagnosed with protein C deficiency and has begun daily IV protein C concentrate (Ceprotin®) treatment. She had no pulmonary or cardiac comorbidities upon discharge from the NICU around four months of age. The brain magnetic resonance imaging (MRI) showed nonspecific white matter injury with multiple small subdural bleeds with no known neurological sequela. She also developed right-sided blindness due to retinal detachment.On presentation to the endocrinology clinic, she plotted below one percentile in the height-for-age and at the third percentile in the weight-for-age charts .She was in no acute distress and had normal vital signs. She was developmentally at par for age. Physical examination was notable for the presence of a subcutaneous port-a-cath on the left upper chest for daily protein C concentrate infusion and well-healed small scar tissues on her chest and abdomen. She had no red reflex on the right eye and self-amputated toes on the left foot. There were no organomegaly or any other dysmorphic features. Laboratory showed undetectable insulin-like growth factor-1 (IGF-1) (<15 ng/mL) and low IGF-binding protein-3 (BP-3)  levels, but normal thyroid studies .Over the next two years, she did not show catch-up growth despite improved nutritional status . She faiHerein, we describe a child with congenital protein C deficiency who was later diagnosed with GHD. Congenital protein C deficiency is a rare and severe bleeding disorder, characterized by a hypercoagulable state as a result of homozygous or compound heterozygous mutation of the PROC gene . AffecteGrowth hormone has multiple effects on various tissues and organs in the body. Besides playing a crucial role in linear growth, it also has vital functions in metabolism and cardiovascular physiology . GH reguThe somatomedin hypothesis suggests that GH mediates its functions, including bone growth, mainly through the induction of hepatic IGF1, which activates transmembranous receptors in chondrocytes . HoweverThe current standard approach for the treatment of GHD is daily subcutaneous injection of recombinant rhGH. However, this route of administration presented a challenge in our patient due to the risk of extensive bruising. Per parent's request and after clearance from her hematologist, we agreed to attempt a trial of intravenous administration of rhGH via her port-a-cath with an option to switch to subcutaneous administration if clinical responses were suboptimal. The half-life of exogenous rhGH is estimated to be around 10–15 minutes . JørgensGrowth hormone stimulates hepatic IGF1 production via the activation of transmembranous receptors. Whether the expression of liver GH receptors is altered in patients with protein C deficiency or the administration of protein C concentrate immediately before the rhGH boluses had suppressed the normal hepatic response is not known. There is no standard dose for IV rhGH replacement. Moreover, we did not assess this treatment's direct effects on lipid metabolism, body composition, or cardiovascular markers. These would have provided direct evidence on the metabolic benefits of rhGH treatment on this patient.IV administration may be an alternative route for treating GHD in eligible patients with permanent vascular access if there are challenges or potential side effects of subcutaneous administration. A significant rise in serum IGF1 levels may not be needed to achieve the growth-promoting effect of IV rhGH therapy.