The interferon-regulated host factor hnRNPA0 modulates HIV-1 production by interference with LTR activity, mRNA trafficking, and programmed ribosomal frameshifting.

The interplay between host factors and viral components impacts viral replication efficiency profoundly. Members of the cellular heterogeneous nuclear ribonucleoprotein family (hnRNPs) have been extensively studied as HIV-1 host dependency factors, but whether they play a role in innate immunity is currently unknown. This study aimed to identify hnRNPA0 as a type I interferon (IFN)-repressed host factor in HIV-1-infected cells. Knockdown of hnRNPA0, a situation that mirrors conditions under IFN stimulation, increased LTR activity, export of unspliced HIV-1 mRNA, viral particle production, and thus, increased infectivity. Conversely, hnRNPA0 overexpression primarily reduced plasmid-driven and integrated HIV-1 long terminal repeat (LTR) activity, significantly decreasing total viral mRNA and protein levels. In addition, high levels of hnRNPA0 significantly reduced the HIV-1 programmed ribosomal frameshifting efficiency, resulting in a shift in the HIV-1 p55/p15 ratio. The HIV-1 alternative splice site usage remained largely unaffected by altered hnRNPA0 levels suggesting that the synergistic inhibition of the LTR activity and viral mRNA transcription, as well as impaired ribosomal frameshifting efficiency, are critical factors for efficient HIV-1 replication regulated by hnRNPA0. The pleiotropic dose-dependent effects under high or low hnRNPA0 levels were further confirmed in HIV-1-infected Jurkat cells. Finally, our study revealed that hnRNPA0 levels in PBMCs were lower in therapy-naive HIV-1-infected individuals compared to healthy controls. Our findings highlight a significant role for hnRNPA0 in HIV-1 replication and suggest that its IFN-I-regulated expression levels are critical for viral fitness allowing replication in an antiviral environment.IMPORTANCERNA-binding proteins, in particular, heterogeneous nuclear ribonucleoproteins (hnRNPs), have been extensively studied. Some act as host dependency factors for HIV-1 since they are involved in multiple cellular gene expression processes. Our study revealed hnRNPA0 as an IFN-regulated host factor, that is differently expressed after IFN-I treatment in HIV-1 target cells and lower expressed in therapy-naïve HIV-1-infected individuals. Our findings demonstrate the significant pleiotropic role of hnRNPA0 in viral replication: In high concentrations, hnRNPA0 limits viral replication by negatively regulating Tat-LTR transcription, retaining unspliced mRNA in the nucleus, and significantly impairing programmed ribosomal frameshifting. Low hnRNPA0 levels as observed in IFN-treated THP-1 cells, particularly facilitate HIV LTR activity and unspliced mRNA export, suggesting a role in innate immunity in favor of HIV replication. Understanding the mode of action between hnRNPA0 and HIV-1 gene expression might help to identify novel therapeutically strategies against HIV-1 and other viruses.

Nerve monitoring in endocrine surgery: Practice patterns differ among surgeons for parathyroidectomy and thyroidectomy.

BACKGROUND: It is unknown whether intraoperative nerve monitoring is associated with reduced vocal cord dysfunction after parathyroidectomy. We aimed to investigate intraoperative nerve monitoring use among Collaborative Endocrine Surgery Quality Improvement Program surgeons and factors associated with vocal cord dysfunction after parathyroidectomy. METHODS: Patients who underwent parathyroidectomy included in the Collaborative Endocrine Surgery Quality Improvement Program (2014-2022) were identified. The annual percent change in parathyroidectomies performed with intraoperative nerve monitoring was calculated using joinpoint regression. Multivariable logistic regression was used to compare outcomes between patients undergoing parathyroidectomy with/without intraoperative nerve monitoring. To compare surgeon-specific trends, Collaborative Endocrine Surgery Quality Improvement Program thyroidectomy and parathyroidectomy datasets (2014-2021) were combined. Parathyroidectomies performed by surgeons who used intraoperative nerve monitoring consistently in thyroidectomy were identified. Factors associated with intraoperative nerve monitoring were examined using multivariable logistic regression. RESULTS: A total of 9,813 patients underwent parathyroidectomy. Intraoperative nerve monitoring was used in 49% of cases (n = 4,818). There was an increase in parathyroidectomies with intraoperative nerve monitoring from 2014 to 2018 (annual percent change 22.2, P = .01), followed by a plateau (2018-2022 annual percent change -0.66, P = .85). Few patients (0.44%, n = 43) developed vocal cord dysfunction. Vocal cord dysfunction was not associated with intraoperative nerve monitoring (adjusted odds ratio 0.92, P = .75). Whereas 41% (n = 56/138) of surgeons used intraoperative nerve monitoring routinely in parathyroidectomy, 65% (n = 90/138) used it routinely in thyroidectomy. Among surgeons who used intraoperative nerve monitoring routinely in thyroidectomy, only 57% used it routinely in parathyroidectomy; factors associated with intraoperative nerve monitoring during parathyroidectomy included reoperation (adjusted odds ratio 2.51, P < .01), secondary/tertiary hyperparathyroidism (adjusted odds ratio 1.42, P = .02), multiglandular disease (adjusted odds ratio 1.76, P < .001), and non-localized disease (adjusted odds ratio 1.65, P < .001). CONCLUSION: Endocrine surgeons use intraoperative nerve monitoring selectively. Surgeons who routinely use intraoperative nerve monitoring during thyroidectomy are more likely to use it during parathyroidectomy. Future studies should determine who may benefit most from intraoperative nerve monitoring in parathyroidectomy.

Enhanced recovery after surgery (ERAS) in colorectal surgery: implementation is still beneficial despite modern surgical and anesthetic care.

PURPOSE: Enhanced recovery after surgery (ERAS) protocols have shown beneficial outcomes in the last 20 years. Nevertheless, simultaneously implemented technical improvements such as minimally invasive access or modified anesthesia care may play a crucial role in optimizing patient outcome. The aim of the study was to investigate the effect of ERAS implementation in a highly specialized colorectal center. METHODS: This is a propensity score matched single-center study comparing the short-term outcomes of patients undergoing elective colorectal surgery in a society-indepedent ERAS program from January 2021 to August 2022 to standard perioperative care from January 2019 to December 2020. RESULTS: Four hundred fifty-six patients were included in the propensity score matched analysis with 228 patients per group (ERAS vs. standard care). Minimally invasive access was used in 80.2% vs. 77.6% (p = 0.88), and there were 16.6% vs. 18.8% (p = 0.92) rectal procedures in the ERAS and standard care group, respectively. Major complications occurred in 10.1% vs. 11.4% (p = 0.65) and anastomotic leakage demanding operative revision in 2.2% vs. 2.6% (p = 0.68) in the ERAS and standard care group, respectively. ERAS lead to a lower number of non-surgical complications compared to standard care (57 vs. 79; p = 0.02). Mean length of stay (LOS) and mean costs per case were lower in ERAS compared to standard care (9.2 ± 5.6 days vs. 12.7 ± 7.4 days, p < 0.01; costs 33,727 ± 15,883 USD vs. 40,309 ± 29,738 USD, p < 0.01). CONCLUSION: The implementation of an ERAS protocol may lead to a reduction of LOS, costs, and a lower number of non-surgical complications even in a highly specialized colorectal unit using modern surgical and anesthetic care. ( ClinialTrials.gov number NCT05773248).

High density of CXCL12-positive immune cell infiltration predicts chemosensitivity and recurrence-free survival in ovarian carcinoma.

BACKGROUND: Ovarian carcinoma is the most lethal gynecologic malignancy because of its late diagnosis, extremely high recurrence rate, and limited curative treatment options. In clinical practice, high-grade serous carcinoma (HGSC) predominates due to its frequency, high aggressiveness, and rapid development of drug resistance. Recent evidence suggests that CXCL12 is an important immunological factor in ovarian cancer progression. Therefore, we investigated the predictive and prognostic significance of the expression of this chemokine in tumor and immune cells in patients with HGSC. METHODS: We studied a cohort of 47 primary high-grade serous ovarian carcinomas and their associated recurrences. A tissue microarray was constructed to evaluate the CXCL12 immunostained tumor tissue. CXCL12 expression was evaluated and statistically analyzed to correlate clinicopathologic data, overall survival, and recurrence-free survival. RESULTS: A high proportion of CXCL12 + positive immune cells in primary ovarian serous carcinoma correlated significantly with chemosensitivity (p = 0.005), overall survival (p = 0.021), and longer recurrence-free survival (p = 0.038). In recurrent disease, high expression of CXCL12 was also correlated with better overall survival (p = 0.040). Univariate and multivariate analysis revealed that high CXCL12 + tumor-infiltrating immune cells (TICs) (HR 0.99, p = 0.042, HR 0.99, p = 0.023, respectively) and combined CXCL12 + /CD66b + infiltration (HR 0.15, p = 0.001, HR 0.13, p = 0.001, respectively) are independent favorable predictive markers for recurrence-free survival. CONCLUSION: A high density of CXCL12 + TICs predicts a good response to chemotherapy, leading to a better overall survival and a longer recurrence-free interval. Moreover, with concomitant high CXCL12/CD66b TIC density, it is an independent favorable predictor of recurrence-free survival in patients with ovarian carcinoma.

Low serum iPTH at the end of surgery is the earliest predictor of postoperative hypocalcemia after total thyroidectomy.

BACKGROUND: The most frequent complication of total thyroidectomy remains hypocalcemia due to low postoperative levels of serum intact parathyroid hormone (iPTH). The purpose of this study was to investigate the role of decreased iPTH at the end of surgery in predicting hypocalcemia. In addition, we examined the percentage decrease of iPTH as potential indicator of hypocalcemia. METHODS: We retrospectively collected the data of patients who underwent total thyroidectomy for benign and malignant diseases at our institution between 2010 and 2022. The iPTH level was measured before and at the end of surgery, and serum calcium levels on the first postoperative day. Demographic, clinical, and biochemical characteristics of patients with low iPTH were compared with patients with normal iPTH levels using ANOVA for continuous variables and χ2-tests for categorical variables. Multivariable logistic regression analysis evaluated the association of iPTH at the end of surgery and the relative reduction of iPTH with postoperative hypocalcemia. RESULTS: The mean age of the 607 patients in this study was 55.6 years, and the female-to-male ratio was 5:1. Goiter was the most common indication for surgery (N = 382, 62.9%), followed by Graves' disease (N = 135, 22.2%). The mean preoperative iPTH was 49.0 pg/ml, while the mean postoperative iPTH was 29.3 pg/ml. A total of 197 patients (32.5%) had an iPTH level below normal, 77 patients (39%), had iPTH levels of 10-15.0 pg/ml and 120 patients (61%) of < 10.0 pg/ml at the end of surgery. Among all patients, 124 (20.4%) developed hypocalcemia on the first postoperative day. The mean percentage of decrease of iPTH was highest among patients with iPTH < 10 pg/ml (76.9%, p < 0.01); this group of patients had also the highest rate of postoperative hypocalcemia on day one (45.0% vs. 26.0% vs 12.2%, p < 0.01). CONCLUSIONS: Measurement of iPTH at the end of total thyroidectomy predicts patients who are at risk for postoperative hypocalcemia. The combination of low serum iPTH with a decrease in iPTH level of ≥ 50% may improve prediction of hypocalcemia compared to iPTH levels alone allowing for early calcium substitution in these patients at high risk of developing postoperative hypocalcemia.

The VBNC state: a fundamental survival strategy of Acinetobacter baumannii.

IMPORTANCE: Currently, the viable but non-culturable (VBNC) state is an underappreciated niche for pathogenic bacteria which provides a continuous source for recurrent infections and transmission. We propose the VBNC state to be a global persistence mechanism used by various A. baumannii strains to cope with many stresses it is confronted with in the clinical environment and in the host. This requires a novel strategy to detect viable cells of this pathogen that is not only based on plating assays.

Using a stress reservoir to improve urine leakage after artificial urinary sphincter implantation.

INTRODUCTION: Although the longest efficacy record, some patients report about urinary leakage during higher intra-abdominal pressure after Artificial Urinary Sphincter (AUS) implantation. To improve the continent results, we placed in addition to the occluding cuff and the pressure regulating balloon, in a second procedure a stress-relief reservoir in lower abdomen, for additional passive pressure transmission to the occluding cuff. METHODS: In this retrospective monocentric data analysis between 2011 and 2018, 80 patients with persistence incontinence after AUS implantation were included. Stress-relief reservoir was indicated in 12 patients with involuntary leakage of urine, that occurred when intra-abdominal pressure raised. RESULTS: In all 12 cases, the stress reservoir was easily implanted and there were no intraoperative complications. In a mean follow-up time of 53 months, the pad per day usage (p/d) improved from 3 (± 1.2) to 1.7 (± 1.5) (p = 0.001). Two patients with multiple previous abdominal surgeries used an equal number of pads after SRR; however, an improvement during physical exertion was reported. Continence situation was evaluated with a questionnaire and was rated as "excellent", "good", or "satisfactory" by 11 (92%) patients. CONCLUSION: The persistence of urinary incontinence after AUS is a challenging topic. Implantation of a stress reservoir in carefully selected patients with urinary leakage during higher intra-abdominal pressure is minimally invasive and offers new options to improve the proven long-term record of AUS. Certainly, more investigations are needed to determine the clinical relevance of this approach.

Routine use of intraoperative nerve monitoring is associated with a reduced risk of vocal cord dysfunction after thyroid cancer surgery.

BACKGROUND: The aim of this study was to investigate the associations between individual surgeon's intraoperative nerve monitoring (IONM) practice and factors associated with vocal cord (VC) dysfunction in patients with thyroid cancer undergoing thyroidectomy. METHODS: Using Collaborative Endocrine Surgery Quality Improvement Program (CESQIP) 2014-21 data, multivariable logistic regression analyses investigated variables associated with short- and long-term VC-dysfunction, associations of routine use of IONM with postoperative outcomes, and patient characteristics associated with IONM use. RESULTS: Among 5,446 patients (76.7% female, mean age 49 years), 68.5% had surgery by surgeons using IONM in ≥ 90% of cases (63% of surgeons, n = 73). Post-operative VC-dysfunction was diagnosed by laryngoscopy in 3.0% of patients in the short-term and 2.7% in the long-term. When surgeons routinely used IONM, the incidence of VC-dysfunction was 2.4% in the short-term and 2.2% in the long-term, compared to 4.4% and 3.7%, respectively, when surgeons did not routinely use IONM (p < 0.01). After adjustment, routine use of IONM was independently associated with reduced risk of short- (OR 0.48, p < 0.01) and long-term (OR 0.52, p < 0.01) VC-dysfunction, a lower risk of postoperative hypoparathyroidism in the short- (OR 0.67, p < 0.01) and long-term (OR 0.54, p < 0.01), and higher likelihood of same-day discharge (OR 2.03, p < 0.01). Extrathyroidal tumor extension and N1-stage were factors associated with postoperative VC-dysfunction in the short- (OR 3.12, p < 0.01; OR 1.92, p = 0.01, respectively) and long-term (OR 3.11, p < 0.01; OR 2.32, p < 0.01, respectively). CONCLUSION: Routine use of IONM was independently associated with a lower risk of endocrine surgery-specific complications and greater likelihood of same-day discharge.

Interlaboratory comparison using inactivated SARS-CoV-2 variants as a feasible tool for quality control in COVID-19 wastewater monitoring.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results. In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values. All PCR-laboratories reported SARS-CoV-2 genome copy equivalents (GCE) for all spiked samples with a mean intra- and inter-laboratory variability of 19 % and 104 %, respectively, largely reproducing the spike-in scheme. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/ml wastewater and had limited predictive value when analyzing at a genome coverage below 60 %. This interlaboratory test demonstrates that despite highly heterogeneous isolation and analysis procedures, overall SARS-CoV-2 GCE and mutations were determined accurately. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection.

Hypoxia-altered cholesterol homeostasis enhances the expression of interferon-stimulated genes upon SARS-CoV-2 infections in monocytes.

Hypoxia contributes to numerous pathophysiological conditions including inflammation-associated diseases. We characterized the impact of hypoxia on the immunometabolic cross-talk between cholesterol and interferon (IFN) responses. Specifically, hypoxia reduced cholesterol biosynthesis flux and provoked a compensatory activation of sterol regulatory element-binding protein 2 (SREBP2) in monocytes. Concomitantly, a broad range of interferon-stimulated genes (ISGs) increased under hypoxia in the absence of an inflammatory stimulus. While changes in cholesterol biosynthesis intermediates and SREBP2 activity did not contribute to hypoxic ISG induction, intracellular cholesterol distribution appeared critical to enhance hypoxic expression of chemokine ISGs. Importantly, hypoxia further boosted chemokine ISG expression in monocytes upon infection with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Mechanistically, hypoxia sensitized toll-like receptor 4 (TLR4) signaling to activation by SARS-CoV-2 spike protein, which emerged as a major signaling hub to enhance chemokine ISG induction following SARS-CoV-2 infection of hypoxic monocytes. These data depict a hypoxia-regulated immunometabolic mechanism with implications for the development of systemic inflammatory responses in severe cases of coronavirus disease-2019 (COVID-19).

Comparison of the Ct-values for genomic and subgenomic SARS-CoV-2 RNA reveals limited predictive value for the presence of replication competent virus.

SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19. In addition to the full length positive-sensed, single-stranded genomic RNA (gRNA), viral subgenomic RNAs (sgRNAs) that are required for expression of the 3' region of the genome are synthesized in virus-infected cells. However, whether these sgRNA-species might be used as a measure of active virus replication and to predict infectivity is still under debate. The commonly used methods to monitor and quantitate SARS-CoV-2 infections are based on RT-qPCR analysis and the detection of gRNA. The infectivity of a sample obtained from nasopharyngeal or throat swabs is associated with the viral load and inversely correlates with Ct-values, however, a cut-off value predicting the infectivity highly depends on the performance of the assay. Furthermore, gRNA derived Ct-values result from nucleic acid detection and do not necessarily correspond to active replicating virus. We established a multiplex RT-qPCR assay on the cobas 6800 omni utility channel concomitantly detecting SARS-CoV-2 gRNAOrf1a/b, sgRNAE,7a,N, and human RNaseP-mRNA used as human input control. We compared the target specific Ct-values with the viral culture frequency and performed ROC curve analysis to determine the assay sensitivity and specificity. We found no advantage in the prediction of viral culture when using sgRNA detection compared to gRNA only, since Ct-values for gRNA and sgRNA were highly correlated and gRNA offered a slightly more reliable predictive value. Single Ct-values alone only provide a very limited prediction for the presence of replication competent virus. Hence, careful consideration of the medical history including symptom onset has to be considered for risk stratification.

Misinformation Caused Increased Urban Mobility and the End of Social Confinement Before the Second Wave of COVID-19 in Amazonia.

Tendentious projections about COVID-19 in Brazil provided an appealing excuse for individuals and decision-makers to justify poor choices during a critical phase of the pandemic. The erroneous results likely contributed to premature resumption of in-person school classes and easing of restrictions on social contact, favoring the resurgence of COVID-19. In Manaus, the largest city in the Amazon region, the COVID-19 pandemic did not end in 2020 of its own accord, but rather rebounded in a disastrous second wave of the disease.

Disease-Specific Survival Trends for Patients Presenting with Differentiated Thyroid Cancer and Distant Metastases in the United States, 1992-2018.

Objective: Differentiated thyroid cancer (DTC) is associated with an excellent prognosis, but patients with distant metastatic DTC have a 10-year disease-specific survival (DSS) of just 50%. The incidence of distant metastatic DTC has steadily increased in the United States since the 1980s. The aim of this study was to examine trends in survival and treatment for patients with distant metastatic DTC. Methods: In this population-based, retrospective cohort study, patients with distant metastatic DTC were identified from the Surveillance, Epidemiology, and End Results-13 cancer registry program. Multivariable logistic and Cox regression analyses were used to examine factors associated with DSS and management. Annual percentage changes in treatment patterns were calculated using log-linear regression. Results: During 1992-2018, 1991 patients (69.7% white, 58.0% female, 47.5% aged ≥65 years) were diagnosed with distant metastatic DTC. Papillary thyroid cancer was the most common histological type (74.5%). While the 10-year DSS for overall DTC increased over time (95.4% for patients diagnosed in 1992-1998, 96.6% in 1999-2008, and 97.3% in 2009-2018; p < 0.01), 10-year DSS for DTC with distant metastases did not change (50.2%, 47.3%, and 52.4%, respectively; p = 0.48). Ten-year DSS rates were reduced for patients aged ≥65 years (28.1%), patients undergoing nonsurgical treatment with external beam radiation therapy and/or systemic therapy (6.0%), and patients undergoing no/unknown treatment (32.8%). On multivariable analysis, oncocytic carcinoma, age 65-79 and ≥80 years, male sex, node-positive disease, larger tumor size, nonsurgical treatment, and no/unknown treatment were associated with increased risk of thyroid cancer death. Between 1992 and 2018, the rate of nonsurgical treatment increased, on average, 1.3% per year (1992-1998: 22.9% vs. 2009-2018: 25.6%; p = 0.03), and the rate of patients receiving no/unknown treatment increased 1.9% per year (1992-1998: 11.3% vs. 2009-2018: 15.6%; p = 0.01). Patients aged 65-79 and ≥80 years were more likely than younger patients to receive nonsurgical management or no/unknown treatment. Conclusion: Patients diagnosed with distant metastatic DTC have experienced no improvement in DSS over the past three decades. An increasing proportion of patients diagnosed with distant metastatic DTC are receiving nonsurgical treatment or no/unknown treatment over time; the proportion was highest among the oldest patients.

Misinformation caused increased urban mobility and the end of social confinement before the second wave of COVID-19 in Amazonia

Tendentious projections about COVID-19 in Brazil provided an appealing excuse for individuals and decision-makers to justify poor choices during a critical phase of the pandemic. The erroneous results likely contributed to premature resumption of in-person school classes and easing of restrictions on social contact, favoring the resurgence of COVID-19. In Manaus, the largest city in the Amazon region, the COVID-19 pandemic did not end in 2020 of its own accord, but rather rebounded in a disastrous second wave of the disease.

SRSF1 acts as an IFN-I-regulated cellular dependency factor decisively affecting HIV-1 post-integration steps.

Efficient HIV-1 replication depends on balanced levels of host cell components including cellular splicing factors as the family of serine/arginine-rich splicing factors (SRSF, 1-10). Type I interferons (IFN-I) play a crucial role in the innate immunity against HIV-1 by inducing the expression of IFN-stimulated genes (ISGs) including potent host restriction factors. The less well known IFN-repressed genes (IRepGs) might additionally affect viral replication by downregulating host dependency factors that are essential for the viral life cycle; however, so far, the knowledge about IRepGs involved in HIV-1 infection is very limited. In this work, we could demonstrate that HIV-1 infection and the associated ISG induction correlated with low SRSF1 levels in intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) during acute and chronic HIV-1 infection. In HIV-1-susceptible cell lines as well as primary monocyte-derived macrophages (MDMs), expression levels of SRSF1 were transiently repressed upon treatment with specific IFNα subtypes in vitro. Mechanically, 4sU labeling of newly transcribed mRNAs revealed that IFN-mediated SRSF1 repression is regulated on early RNA level. SRSF1 knockdown led to an increase in total viral RNA levels, but the relative proportion of the HIV-1 viral infectivity factor (Vif) coding transcripts, which is essential to counteract APOBEC3G-mediated host restriction, was significantly reduced. In the presence of high APOBEC3G levels, however, increased LTR activity upon SRSF1 knockdown facilitated the overall replication, despite decreased vif mRNA levels. In contrast, SRSF1 overexpression significantly impaired HIV-1 post-integration steps including LTR transcription, alternative splice site usage, and virus particle production. Since balanced SRSF1 levels are crucial for efficient viral replication, our data highlight the so far undescribed role of SRSF1 acting as an IFN-modulated cellular dependency factor decisively regulating HIV-1 post-integration steps.

The prognostic significance of CXCR4 and SDF-1 in differentiated thyroid cancer depends on CD8+ density.

BACKGROUND: Tumor infiltration with cytotoxic CD8+ T-cells is associated with a favorable outcome in several neoplasms, including thyroid cancer. The chemokine axis CXCR4/SDF-1 correlates with more aggressive tumors, but little is known concerning the prognostic relevance in relation to the tumor immune microenvironment of differentiated thyroid cancer (DTC). METHODS: A tissue microarray (TMA) of 37 tumor specimens of primary DTC was analyzed by immunohistochemistry (IHC) for the expression of CD8+, CXCR4, phosphorylated CXCR4 and SDF-1. A survival analysis was performed on a larger collective (n = 456) at RNA level using data from The Cancer Genome Atlas (TCGA) papillary thyroid cancer cohort. RESULTS: Among the 37 patients in the TMA-cohort, the density of CD8+ was higher in patients with less advanced primary tumors (median cells/TMA-punch: 12.5 (IQR: 6.5, 12.5) in T1-2 tumors vs. 5 (IQR: 3, 8) in T3-4 tumors, p = 0.05). In the TCGA-cohort, CXCR4 expression was higher in patients with cervical lymph node metastasis compared to N0 or Nx stage (CXCR4high/low 116/78 vs. 97/116 vs. 14/35, respectively, p = 0.001). Spearman's correlation analysis of the TMA-cohort demonstrated that SDF-1 was significantly correlated with CXCR4 (r = 0.4, p = 0.01) and pCXCR4 (r = 0.5, p = 0.002). In the TCGA-cohort, density of CD8+ correlated with CXCR4 and SDF-1 expression (r = 0.58, p < 0.001; r = 0.4, p < 0.001). The combined marker analysis of the TCGA cohort demonstrated that high expression of both, CXCR4 and SDF-1 was associated with reduced overall survival in the CD8 negative TCGA cohort (p = 0.004). CONCLUSION: These findings suggest that the prognostic significance of CXCR4 and SDF-1 in differentiated thyroid cancer depends on the density of CD8 positive T-lymphocytes. Further studies with larger sample sizes are needed to support our findings and inform future investigations of new treatment and diagnostic options for a more personalized approach for patients with differentiated thyroid cancer.

NVX-CoV2373-induced cellular and humoral immunity towards parental SARS-CoV-2 and VOCs compared to BNT162b2 and mRNA-1273-regimens.

BACKGROUND: The NVX-CoV2373-vaccine has recently been licensed, although knowledge on vaccine-induced humoral and cellular immunity towards the parental strain and variants of concern (VOCs) in comparison to mRNA-regimens is limited. METHODS: In this observational study, 66 individuals were recruited to compare immunogenicity and reactogenicity of NVX-CoV2373 with BNT162b2 or mRNA-1273. Vaccine-induced antibodies were analyzed using ELISA and neutralization assays, specific CD4 and CD8 T-cells were characterized based on intracellular cytokine staining using flow-cytometry after antigen-specific stimulation with parental spike or VOCs. RESULTS: Two doses of NVX-CoV2373 strongly induced anti-spike IgG, although IgG-levels were lower than after vaccination with BNT162b2 or mRNA-1273 (p = 0.006). Regardless of the vaccine and despite different IgG-levels, neutralizing activity towards VOCs was highest for Delta, followed by BA.2 and BA.1. The protein-based vaccine failed to induce any spike-specific CD8 T-cells which were detectable in 3/22 (14%) individuals only. In contrast, spike-specific CD4 T-cells were induced in 18/22 (82%) individuals, although their levels were lower (p<0.001), had lower CTLA-4 expression (p<0.0001) and comprised less multifunctional cells co-expressing IFNγ, TNFα and IL-2 (p = 0.0007). Unlike neutralizing antibodies, NVX-CoV2373-induced CD4 T-cells equally recognized all tested VOCs from Alpha to Omicron. In individuals with a history of infection, one dose of NVX-CoV2373 had similar immunogenicity as two doses in non-infected individuals. The vaccine was overall well tolerated. CONCLUSION: NVX-CoV2373 strongly induced spike-specific antibodies and CD4 T-cells, albeit at lower levels as mRNA-regimens. Cross-reactivity of CD4 T-cells towards the parental strain and all tested VOCs may hold promise to protect from severe disease.

Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.

Molecular Analyses of Clinical Isolates and Recombinant SARS-CoV-2 Carrying B.1 and B.1.617.2 Spike Mutations Suggest a Potential Role of Non-Spike Mutations in Infection Kinetics.

Some of the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are less susceptible to neutralization with post-vaccine sera and monoclonal antibodies targeting the viral spike glycoprotein. This raises concerns of disease control, transmissibility, and severity. Numerous substitutions have been identified to increase viral fitness within the nucleocapsid and nonstructural proteins, in addition to spike mutations. Therefore, we sought to generate infectious viruses carrying only the variant-specific spike mutations in an identical backbone to evaluate the impact of spike and non-spike mutations in the virus life cycle. We used en passant mutagenesis to generate recombinant viruses carrying spike mutations of B.1 and B.1.617.2 variants using SARS-CoV-2- bacterial artificial chromosome (BAC). Neutralization assays using clinical sera yielded comparable results between recombinant viruses and corresponding clinical isolates. Non-spike mutations for both variants neither seemed to effect neutralization efficiencies with monoclonal antibodies nor the response to treatment with inhibitors. However, live-cell imaging and microscopy revealed differences, such as persisting syncytia and pronounced cytopathic effect formation, as well as their progression between BAC-derived viruses and clinical isolates in human lung epithelial cell lines and primary bronchial epithelial cells. Complementary RNA analyses further suggested a potential role of non-spike mutations in infection kinetics.

Endocrine surgeons are performing more thyroid lobectomies for low-risk differentiated thyroid cancer since the 2015 ATA guidelines.

BACKGROUND: The 2015 American Thyroid Association guidelines recommended either total thyroidectomy or lobectomy for surgical treatment of low-risk differentiated thyroid cancer and de-escalated recommendations for central neck dissections. The study aim was to investigate how practice patterns among endocrine surgeons have changed over time. METHODS: All adult patients with low-risk differentiated thyroid cancers (T1-T2, N0/Nx, M0/Mx) in the Collaborative Endocrine Surgery Quality Improvement Program (2014-2021) were identified. The outcomes between patients undergoing lobectomy versus total thyroidectomy were compared using multivariable logistic regression. The annual percent change in the proportion of lobectomies and central neck dissections performed was estimated using joinpoint regression. RESULTS: In total, 5,567 patients with low-risk differentiated thyroid cancers were identified. Of these, 2,261 (40.6%) were very low-risk tumors ≤1 cm, and 2,983 (53.6%) were low-risk tumors >1 and <4 cm. Most patients (67.9%) underwent total thyroidectomy. Compared to total thyroidectomy, lobectomy was associated with outpatient surgery (adjusted odds ratio 5.19, P < .001), a decreased risk of postoperative emergency department visits (adjusted odds ratio 0.63, P = .03), and decreased risk of hypoparathyroidism events (adjusted odds ratio 0.03, P < .001). Compared to before (2014-2015), patients undergoing surgery after publication of the revised guidelines (2016-2021) had higher odds of lobectomy and lower odds of central neck dissection for tumors ≤1 cm (lobectomy adjusted odds ratio 2.70, P < .001; central neck dissections adjusted odds ratio 0.64, P = .03) and tumors between 1 and 4 cm (lobectomy adjusted odds ratio 2.27, P < .001; central neck dissection adjusted odds ratio 0.62, P < .001). CONCLUSION: After publication of the 2015 American Thyroid Association guidelines, there has been an increase in thyroid lobectomies as a proportion of all thyroid operations performed by endocrine surgeons for low-risk differentiated thyroid cancer. This has implications for reduced health care use and costs, with potential population-level benefits.