High-resolution cryo-EM of the human CDK-activating kinase for structure-based drug design.

Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.

Decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA in raw sewage from university dormitories.

Introduction: Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA has been frequently detected in sewage from many university dormitories to inform public health decisions during the COVID-19 pandemic, a clear understanding of SARS-CoV-2 RNA persistence in site-specific raw sewage is still lacking. To investigate the SARS-CoV-2 RNA persistence, a field trial was conducted in the University of Tennessee dormitories raw sewage, similar to municipal wastewater. Methods: The decay of enveloped SARS-CoV-2 RNA and non-enveloped Pepper mild mottle virus (PMMoV) RNA was investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in raw sewage at 4°C and 20°C. Results: Temperature, followed by the concentration level of SARS-CoV-2 RNA, was the most significant factors that influenced the first-order decay rate constants (k) of SARS-CoV-2 RNA. The mean k values of SARS-CoV-2 RNA were 0.094 day-1 at 4°C and 0.261 day-1 at 20°C. At high-, medium-, and low-concentration levels of SARS-CoV-2 RNA, the mean k values were 0.367, 0.169, and 0.091 day-1, respectively. Furthermore, there was a statistical difference between the decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA at different temperature conditions. Discussion: The first decay rates for both temperatures were statistically comparable for SARS-CoV-2 RNA, which showed sensitivity to elevated temperatures but not for PMMoV RNA. This study provides evidence for the persistence of viral RNA in site-specific raw sewage at different temperature conditions and concentration levels.

The CDK7 inhibitor CT7001 (Samuraciclib) targets proliferation pathways to inhibit advanced prostate cancer.

BACKGROUND: Current strategies to inhibit androgen receptor (AR) are circumvented in castration-resistant prostate cancer (CRPC). Cyclin-dependent kinase 7 (CDK7) promotes AR signalling, in addition to established roles in cell cycle and global transcription, providing a rationale for its therapeutic targeting in CRPC. METHODS: The antitumour activity of CT7001, an orally bioavailable CDK7 inhibitor, was investigated across CRPC models in vitro and in xenograft models in vivo. Cell-based assays and transcriptomic analyses of treated xenografts were employed to investigate the mechanisms driving CT7001 activity, alone and in combination with the antiandrogen enzalutamide. RESULTS: CT7001 selectively engages with CDK7 in prostate cancer cells, causing inhibition of proliferation and cell cycle arrest. Activation of p53, induction of apoptosis, and suppression of transcription mediated by full-length and constitutively active AR splice variants contribute to antitumour efficacy in vitro. Oral administration of CT7001 represses growth of CRPC xenografts and significantly augments growth inhibition achieved by enzalutamide. Transcriptome analyses of treated xenografts indicate cell cycle and AR inhibition as the mode of action of CT7001 in vivo. CONCLUSIONS: This study supports CDK7 inhibition as a strategy to target deregulated cell proliferation and demonstrates CT7001 is a promising CRPC therapeutic, alone or in combination with AR-targeting compounds.

SARS-CoV-2 raw wastewater surveillance from student residences on an urban university campus.

The COVID-19 pandemic brought about an urgent need to monitor the community prevalence of infection and detect the presence of SARS-CoV-2. Testing individual people is the most reliable method to measure the spread of the virus in any given community, but it is also the most expensive and time-consuming. Wastewater-based epidemiology (WBE) has been used since the 1960s when scientists implemented monitoring to measure the effectiveness of the Polio vaccine. Since then, WBE has been used to monitor populations for various pathogens, drugs, and pollutants. In August 2020, the University of Tennessee-Knoxville implemented a SARS-CoV-2 surveillance program that began with raw wastewater surveillance of the student residence buildings on campus, the results of which were shared with another lab group on campus that oversaw the pooled saliva testing of students. Sample collection began at 8 am, and the final RT-qPCR results were obtained by midnight. The previous day's results were presented to the campus administrators and the Student Health Center at 8 am the following morning. The buildings surveyed included all campus dormitories, fraternities, and sororities, 46 buildings in all representing an on-campus community of over 8,000 students. The WBE surveillance relied upon early morning "grab" samples and 24-h composite sampling. Because we only had three Hach AS950 Portable Peristaltic Sampler units, we reserved 24-h composite sampling for the dormitories with the highest population of students. Samples were pasteurized, and heavy sediment was centrifuged and filtered out, followed by a virus concentration step before RNA extraction. Each sample was tested by RT-qPCR for the presence of SARS-CoV-2, using the CDC primers for N Capsid targets N1 and N3. The subsequent pooled saliva tests from sections of each building allowed lower costs and minimized the total number of individual verification tests that needed to be analyzed by the Student Health Center. Our WBE results matched the trend of the on-campus cases reported by the student health center. The highest concentration of genomic copies detected in one sample was 5.06 × 107 copies/L. Raw wastewater-based epidemiology is an efficient, economical, fast, and non-invasive method to monitor a large community for a single pathogen or multiple pathogen targets.

'Diagnostic downshift': clinical and system consequences of extrapolating secondary care testing tactics to primary care.

Numerous drivers push specialist diagnostic approaches down to primary care ('diagnostic downshift'), intuitively welcomed by clinicians and patients. However, primary care's different population and processes result in under-recognised, unintended consequences. Testing performs poorer in primary care, with indication creep due to earlier, more undifferentiated presentation and reduced accuracy due to spectrum bias and the 'false-positive paradox'. In low-prevalence settings, tests without near-100% specificity have their useful yield eclipsed by greater incidental or false-positive findings. Ensuing cascades and multiplier effects can generate clinician workload, patient anxiety, further low-value tests, referrals, treatments and a potentially nocebic population 'disease' burden of unclear benefit. Increased diagnostics earlier in pathways can burden patients and stretch general practice (GP) workloads, inducing downstream service utilisation and unintended 'market failure' effects. Evidence is tenuous for reducing secondary care referrals, providing patient reassurance or meaningfully improving clinical outcomes. Subsequently, inflated investment in per capita testing, at a lower level in a healthcare system, may deliver diminishing or even negative economic returns. Test cost poorly represents 'value', neglecting under-recognised downstream consequences, which must be balanced against therapeutic yield. With lower positive predictive values, more tests are required per true diagnosis and cost-effectiveness is rarely robust. With fixed secondary care capacity, novel primary care testing is an added cost pressure, rarely reducing hospital activity. GP testing strategies require real-world evaluation, in primary care populations, of all downstream consequences. Test formularies should be scrutinised in view of the setting of care, with interventions to focus rational testing towards those with higher pretest probabilities, while improving interpretation and communication of results.

Coding-Complete Genome Sequence of a SARS-CoV-2 Variant Obtained from Raw Sewage at the University of Tennessee-Knoxville Campus.

Reported here is a coding-complete genome sequence of a SARS-CoV-2 variant obtained from raw wastewater samples at the University of Tennessee-Knoxville campus. This sequence provides insight into SARS-CoV-2 variants that circulate on large college campuses but remain mostly undetected.

Quantification of the Role of Chemical Desorption in Molecular Clouds.

ConspectusDark molecular clouds have low temperatures of approximately 10 K and experience very little UV irradiation. These clouds are the birthplace of new stars and consist of gas and dust particles. The latter can act as a meeting place to facilitate surface chemistry to form saturated molecules such as formaldehyde, methyl formate, and dimethyl ether. These complex organic molecules or COMs become encapsulated in the ice that forms on the dust grains, and these ices are the precursor for cometary ices and other icy bodies. They likely played a role in bringing material to the early earth.Although these COMs are likely formed on the surfaces of dust grains, several of them have been detected in the gas phase. This means that they have desorbed from the grain under these cold, dark conditions where thermal desorption and photodesorption are negligible. It has been speculated that reactive, or chemical, desorption is responsible for the high gas-phase abundance. After a surface reaction, its products might be vibrationally, translationally, and/or rotationally excited. Dissipation of the excess energy to translational energy can briefly increase the desorption rate, leading to chemical desorption. Astrochemical modellers have added terms to their rate equations to account for this effect. These terms, however, have had little experimental or theoretical verification.In this Account, we use classical molecular dynamics (MD) simulations to give adsorbed molecules a fixed amount of energy as a proxy for excess energy and to record whether this leads to desorption. The excitation energy can be varied freely while keeping all other variables constant. This allows for the study of trends rather than being limited to a single reaction and a single system. The focus is on the dependence of the chemical desorption on the excitation energy, excitation type, and binding energy. Rotational and vibrational excitation was explicitly taken into account. An analytical expression for the chemical desorption probability was obtained in this way. It depends on the binding energy and reaction enthalpy. This expression was then implemented in a gas-grain astrochemical code to simulate the chemical evolution of a dark molecular cloud, and the results were compared against observational abundances of COMs in three different molecular clouds. The results with our new expression based on the MD simulations show good agreement for all species except H2CO, which has both gas-phase and surface-formation routes. This is a significant improvement over models without chemical desorption or with other expressions for chemical desorption, as frequently used by other authors. It is encouraging to see that a general description with a firmer theoretical basis leads to a significant improvement. Understanding chemical desorption can help to explain the unexpectedly high gas-phase abundance of some COMs, and chemical desorption also provides a link between the gas phase and the ice mantle, and its understanding might help in creating a diagnostic tool to learn more about the ice composition.

Living risk prediction algorithm (QCOVID) for risk of hospital admission and mortality from coronavirus 19 in adults: national derivation and validation cohort study.

OBJECTIVE: To derive and validate a risk prediction algorithm to estimate hospital admission and mortality outcomes from coronavirus disease 2019 (covid-19) in adults. DESIGN: Population based cohort study. SETTING AND PARTICIPANTS: QResearch database, comprising 1205 general practices in England with linkage to covid-19 test results, Hospital Episode Statistics, and death registry data. 6.08 million adults aged 19-100 years were included in the derivation dataset and 2.17 million in the validation dataset. The derivation and first validation cohort period was 24 January 2020 to 30 April 2020. The second temporal validation cohort covered the period 1 May 2020 to 30 June 2020. MAIN OUTCOME MEASURES: The primary outcome was time to death from covid-19, defined as death due to confirmed or suspected covid-19 as per the death certification or death occurring in a person with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the period 24 January to 30 April 2020. The secondary outcome was time to hospital admission with confirmed SARS-CoV-2 infection. Models were fitted in the derivation cohort to derive risk equations using a range of predictor variables. Performance, including measures of discrimination and calibration, was evaluated in each validation time period. RESULTS: 4384 deaths from covid-19 occurred in the derivation cohort during follow-up and 1722 in the first validation cohort period and 621 in the second validation cohort period. The final risk algorithms included age, ethnicity, deprivation, body mass index, and a range of comorbidities. The algorithm had good calibration in the first validation cohort. For deaths from covid-19 in men, it explained 73.1% (95% confidence interval 71.9% to 74.3%) of the variation in time to death (R2); the D statistic was 3.37 (95% confidence interval 3.27 to 3.47), and Harrell's C was 0.928 (0.919 to 0.938). Similar results were obtained for women, for both outcomes, and in both time periods. In the top 5% of patients with the highest predicted risks of death, the sensitivity for identifying deaths within 97 days was 75.7%. People in the top 20% of predicted risk of death accounted for 94% of all deaths from covid-19. CONCLUSION: The QCOVID population based risk algorithm performed well, showing very high levels of discrimination for deaths and hospital admissions due to covid-19. The absolute risks presented, however, will change over time in line with the prevailing SARS-C0V-2 infection rate and the extent of social distancing measures in place, so they should be interpreted with caution. The model can be recalibrated for different time periods, however, and has the potential to be dynamically updated as the pandemic evolves.

Can displaced extra-articular fractures of the scapula be stabilized through a direct lateral-column approach.

BACKGROUND: Displaced extraarticular fractures of the scapula are uncommon, and rarely require operative fixation. When managed operatively, a posterior Judet approach with detachment of the deltoid muscle from the scapular spine and elevation of the infraspinatus from its fossa, is often performed. This approach is invasive and involves extensive soft tissue dissection. This paper describes the utility of single-column fixation with a direct lateral column approach, with mobilisation of the deltoid, and elevation of the interval between the infraspinatus and teres minor. Our aim is to assess the functional and radiological outcomes of this alternative approach. METHODS: We performed a retrospective cohort study of all patients who underwent operative fixation of their extra-articular scapula fracture (with at least 12 months follow up), using the direct lateral-column approach. Operative indications included patients with an extra articular scapular neck or body fracture, with medial/lateral displacement of ≥20 mm, angulation ≥45°, double disruption of the shoulder suspensory complex, and glenopolar angle (GPA) ≤ 22°. Functional outcomes were assessed by the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire, Subjective Shoulder Value (SSV), pain score & return to work. Radiological assessment was done by 2 independent assessors. RESULTS: Between January 2014 and December 2016, 12 patients (11 males and 1 female) underwent fixation of their scapula fracture using this approach. Eleven patients (91.7%) returned their questionnaire at an average of 15.6 months (12-28 months). All fractures had healed at the time of the final follow-up. The scapular neck angulation was corrected from 38.7° pre-operatively (0-74°) to 3.6° post-operatively (0-20°). The mean post-operative GPA was 35.4° (30.2°-42.0°). None of the patients had superficial or deep infections, or post-operative neurovascular injuries. Two patients underwent elective removal of their clavicle hook plates. The mean SSV was 88.9 (70-100) and mean pain score was 1.5 (0-8). The mean DASH score was 11.4 (0-51.6). Ten patients returned back to their pre-injury work, with an average return to work of 3.3 months (2 weeks-8 months). Only one patient had a poor DASH score. He had associated ipsilateral segmental fracture of the humerus, fracture of the lateral clavicle and brachial plexus injury. CONCLUSIONS: The direct lateral-column approach is an alternative technique for fixation of the extra-articular neck and body scapular fracture. Plating of the lateral column of the scapula through this single approach is associated with correction of scapular angular deformity, no complications and good clinical results at more than 12 months' follow-up.

Re-establishing the Role of Robot-assisted Radical Cystectomy After the 2020 EAU Muscle-invasive and Metastatic Bladder Cancer Guideline Panel Recommendations.

The EAU guidelines panel on muscle-invasive and metastatic bladder cancer (MIBC) recently recommended open radical cystectomy (ORC) as the best surgical approach for MIBC patients. We critically re-examine the indications for considering ORC as the first choice over robot-assisted radical cystectomy. To the best of our knowledge, this is not supported by trials or meta-analyses.

Anatomical robotic prostatectomy: technical factors to achieve superb continence and erectile function.

Debate continues as to the superiority of robotic versus open radical prostatectomy for the surgical treatment of localized prostate cancer. Despite this controversy, retrospective data from high volume centres has demonstrated RARP is associated with improved pentafecta outcomes with lower transfusion rates, less incontinence, lower positive surgical margins and improved potency. Advocates of robotic assisted radical prostatectomy (RARP) believe an enhanced visual field, the precision afforded by robotic technology as well as lack of bleeding, sharp dissection and delicate tissue handling lead to improved outcomes. Prostate Cancer is the second most common cancer diagnosed in men, and as the number of post-surgical patients increases, the complications of urinary incontinence and erectile dysfunction not only have a significant negative impact on patients' quality of life, but have become an expanding part of clinical practice. This article outlines what are believed to be the most important strategies based on anatomical knowledge and technical expertise, that allow robotic prostatectomists to achieve superb outcomes in urinary and erectile function.

Defining Prostate Cancer at Favorable Intermediate Risk: The Potential Utility of Magnetic Resonance Imaging and Genomic Tests.

PURPOSE: We determined whether prostate multiparametric magnetic resonance imaging and genomic biomarkers might help further define patients with favorable intermediate risk prostate cancer which could safely be considered suitable for active surveillance. MATERIALS AND METHODS: From our institutional database we identified 509 patients who underwent radical prostatectomy with preoperative magnetic resonance imaging and a postoperative Decipher® prostate cancer test. According to the NCCN® (National Comprehensive Cancer Network®) risk stratification 125 men had favorable intermediate and 171 had unfavorable intermediate risk disease. Univariable and multivariable binary logistic regression analyses were done to test the utility of different variables in predicting adverse pathology, defined as Gleason Grade Group greater than 2, pT3b or pN1. RESULTS: On univariable analysis favorable intermediate risk, multiparametric magnetic resonance imaging and the prostate cancer test significantly predicted adverse pathology. On multivariable analysis favorable intermediate risk and the prostate cancer test maintained independent predictive value while multiparametric magnetic resonance imaging did not meet statistical significance (p = 0.059). The 19 patients at favorable intermediate risk with high genomic risk had an adverse pathology rate slightly higher than patients at unfavorable intermediate risk (42.1% vs 39.8%, p = 0.56). Those at low genomic risk had an adverse pathology rate slightly lower than patients at very low or low risk (7.5% vs 10.2%, p = 0.84). The 31 patients at favorable intermediate risk but at high multiparametric magnetic resonance imaging and genomic risk had an adverse pathology rate slightly lower than patients at unfavorable intermediate risk (25.8% vs 39.8%, p = 0.14). Those at low multiparametric magnetic resonance imaging and genomic risk had an adverse pathology rate slightly lower than patients at very low or low risk (8.5% vs 10.2%, p = 0.89). CONCLUSIONS: Multiparametric magnetic resonance imaging and the Decipher test allowed us to better define the risk of adverse pathology in patients at favorable intermediate risk who were diagnosed with prostate cancer.

Supra-pubic versus urethral catheter after robot-assisted radical prostatectomy: systematic review of current evidence.

PURPOSE: To provide latest evidence on the use of suprapubic catheter (SPC) versus urethral catheter (UC) after robot-assisted laparoscopic radical prostatectomy (RARP). MATERIALS AND METHODS: A systematic revision of literature was performed up to September 2017 using different search engines (Pubmed, Ovid, Scopus) to identified studies comparing the use of SPC versus standard UC after RARP. Identification and selection of the studies were conducted according to the preferred reporting items for systematic reviews and meta-analysis criteria. For continuous outcomes, the weighted mean difference (WMD) was used as a summary measure, whereas the odds ratio (OR) or risk ratio (RR) with 95% confidence interval (CI) was calculated for binary variables. RR was preferred in cases of a high number of events to avoid overestimation. Pooled estimates were calculated using the random-effect model to account for clinical heterogeneity. All statistical analyses were performed using Review manager 5 (Cochrane Collaboration, Oxford, UK). RESULTS: Eight studies were identified and included in this systematic review, namely 3 RCTs, 4 non-randomized prospective studies, and one retrospective study. A total of 966 RARP cases were collected for the cumulative analysis. Among them, 492 patients received standard UC and 474 SPC placement after RARP. UC patients had higher baseline PSA (WMD 0.44 ng/ml; p = 0.02). Visual Analog Scale (VAS) score was found to be significantly lower in patients with SPC at postoperative day 7 (WMD 0.53; 95% CI 0.13-0.93; p = 0.009). Regarding penile pain, a significant difference in favor of the SPC group was found at postoperative day 7 assessment (WMD 1.2; 95% CI 0.82-1.6; p < 0.001). More patients in the SPC group reported "not at all" or "minimal pain" at this time point (OR 0.17, 95% CI 0.06, 0.44; p < 0.001). No significant differences were found in terms of continence recovery rate at 6-12 weeks between the groups (UC 78.7%, 88.2%; RR 0.92, 95% CI 0.84, 1.01; p = 0.09). Similarly, no differences were found in terms of catheter-related issues (p = 0.17). However, UC patients had lower likelihood of overall complications (OR 0.44, 95% CI 0.21-0.89, p = 0.02). CONCLUSIONS: Available evidence suggests that the use of SPC can be a viable option for postoperative urine drainage after RARP, as it can translate into decreased postoperative pain without carrying a significant higher risk of catheter-related complications. Further investigation seems to be warranted, ideally within the framework of a multicentre randomized study with standardized analysis of outcomes.

Use of 3D printer technology to facilitate surgical correction of a complex vascular anomaly with esophageal entrapment in a dog.

A 10 week old female intact Staffordshire terrier was presented with a total of five congenital cardio-thoracic vascular anomalies consisting of a patent ductus arteriosus (PDA) with an aneurysmic dilation, pulmonic stenosis, persistent right aortic arch, aberrant left subclavian artery and persistent left cranial vena cava. These abnormalities were identified with a combination of echocardiogram and computed tomography angiography (CTA). The abnormalities were associated with esophageal entrapment, regurgitation, and volume overload of the left heart with left atrial and ventricular enlargement. A 2 cm diameter aneurysmic dilation at the junction of the PDA, right aortic arch and aberrant left subclavian artery presented an unusual surgical challenge and precluded simple circumferential ligation and transection of the structure. A full scale three dimensional model of the heart and vasculature was constructed from the CTA and plasma sterilized. The model was used preoperatively to facilitate surgical planning and enhance intraoperative communication and coordination between the surgical and anesthesia teams. Intraoperatively the model facilitated spatial orientation, atraumatic vascular dissection, instrument sizing and positioning. A thoracoabdominal stapler was used to close the PDA aneurysm prior to transection. At the four-month postoperative follow-up the patient was doing well. This is the first reported application of new imaging and modeling technology to enhance surgical planning when approaching correction of complex cardiovascular anomalies in a dog.