Refining detection methods for emerging SARS-CoV-2 mutants in wastewater: A case study on the Omicron variants.

COVID-19 is an ongoing public health threat worldwide driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Wastewater surveillance has emerged as a complementary tool to clinical surveillance to control the COVID-19 pandemic. With the emergence of new variants of SARS-CoV-2, accumulated mutations that occurred in the SARS-CoV-2 genome raise new challenges for RT-qPCR diagnosis used in wastewater surveillance. There is a pressing need to develop refined methods for modifying primer/probes to better detect these emerging variants in wastewater. Here, we exemplified this process by focusing on the Omicron variants, for which we have developed and validated a modified detection method. We first modified the primers/probe mismatches of three assays commonly used in wastewater surveillance according to in silico analysis results for the mutations of 882 sequences collected during the fifth-wave outbreak in Hong Kong, and then evaluated them alongside the seven original assays. The results showed that five of seven original assays had better sensitivity for detecting Omicron variants, with the limits of detection (LoDs) ranging from 1.53 to 2.76 copies/µL. UCDC-N1 and Charité-E sets had poor performances, having LoDs higher than 10 copies/µL and false-positive/false-negative results in wastewater testing, probably due to the mismatch and demonstrating the need for modification of primer/probe sequences. The modified assays exhibited higher sensitivity and specificity, along with better reproducibility in detecting 81 wastewater samples. In addition, the sequencing results of six wastewater samples by Illumina also validated the presence of mismatches in the primer/probe binding sites of the three assays. This study highlights the importance of re-configuration of the primer-probe sets and refinements for the sequences to ensure the diagnostic effectiveness of RT-qPCR detection.

Clinical and prognostic features associated with anti-Ro52 autoantibodies in connective tissue diseases patients with interstitial lung disease.

OBJECTIVES: To define the clinical and prognostic features associated with anti-Ro52 autoantibodies in patients with connective tissue diseases with interstitial lung disease (CTD-ILD). METHODS: A total of 238 patients with CTD-ILD were included in this single-centre retrospective cohort study. Patients with positive anti-Ro52 antibodies were selected as the study group, and those with negative anti-Ro52 antibodies were included in the control group. Clinical and follow-up data were analysed. RESULTS: Among 238 patients, 145 (60.92%) were positive for the anti-Ro52 antibody. These patients were more likely to have respiratory symptoms at baseline, with more organising pneumonia (OP) patterns and worse forced vital capacity (FVC). Follow-up data were obtained for ILD progression in 170 patients. Varying degrees of progression in pulmonary function (PF) or imaging were found in 48 patients (28.24%) with CTD-ILD. A dichotomous logistic analysis based on the presence or absence of progress showed no correlation with anti-Ro52 antibodies. During the follow-up of 170 patients, there were 35 deaths: 24 in the anti-Ro52 antibody positive group and 11 in the anti-Ro52 antibody negative group. Kaplan-Meier survival curves were used to describe the difference in survival between the two groups (mortality 17.14% vs. 12.5%, log-rank p=0.287). The multivariate logistic analysis showed that ILD progression was associated with older age, worse FVC and diffusion capacity for carbon monoxide at baseline, higher levels of C-reactive protein, serum ferritin, immunoglobulin G and lower absolute lymphocyte count. CONCLUSIONS: Anti-Ro52 antibodies may predict more severe lung damage in CTD-ILD; however, anti-Ro52 antibodies were not correlated with progression and death in patients with ILD.

QMRA of beach water by Nanopore sequencing-based viability-metagenomics absolute quantification.

The majority of the current regulatory practices for routine monitoring of beach water quality rely on the culture-based enumeration of faecal indicator bacteria (FIB) to develop criteria for promoting the general public's health. To address the limitations of culture methods and the arguable reliability of FIB in indicating health risks, we developed a Nanopore metagenomic sequencing-based viable cell absolute quantification workflow to rapidly and accurately estimate a broad range of microbes in beach waters by a combination of propidium monoazide (PMA) and cellular spike-ins. Using the simple synthetic bacterial communities mixed with viable and heat-killed cells, we observed near-complete relic DNA removal by PMA with minimal disturbance to the composition of viable cells, demonstrating the feasibility of PMA treatment in profiling viable cells by Nanopore sequencing. On a simple mock community comprised of 15 prokaryotic species, our results showed high accordance between the expected and estimated concentrations, suggesting the accuracy of our method in absolute quantification. We then further assessed the accuracy of our method for counting viable Escherichia coli and Vibrio spp. in beach waters by comparing to culture-based method, which were also in high agreement. Furthermore, we demonstrated that 1 Gb sequences obtained within 2 h would be sufficient to quantify a species having a concentration of ≥ 10 cells/mL in beach waters. Using our viability-resolved quantification workflow to assess the microbial risk of the beach water, we conducted (1) screening-level quantitative microbial risk assessment (QMRA) to investigate human illness risk and site-specific risk patterns that might guide risk management efforts and (2) metagenomics-based resistome risk assessment to evaluate another layer of risk caused by difficult illness treatment due to antimicrobial resistance (AMR). In summary, our metagenomic workflow for the rapid absolute quantification of viable bacteria demonstrated its great potential in paving new avenues toward holistic microbial risk assessment.

Sulforaphene targets NLRP3 inflammasome to suppress M1 polarization of macrophages and inflammatory response in rheumatoid arthritis.

This work aimed to explore the therapeutic effect and target of sulforaphene (LF) in mice with rheumatoid arthritis (RA). Lipopolysaccharide (LPS) and IFN-γ were added to induce the M1 polarization of SMG cells, and later cells were pretreated with 5 µM and 15 µM LF. M1 cell proportion was detected by flow cytometry (FCM), inflammatory factors were measured by enzyme-linked immunosorbent assay, and protein levels were analyzed by western blotting (WB) assay. Besides, small molecule-protein docking and pull-down assays were carried out to detect the binding of LF to NLRP3. After the knockdown of NLRP3 in SMG cells, the effect of LF was further detected. The RA mouse model was induced with collagen antibody and LPS, after LF intervention, H&E staining was performed to detect the pathological changes in mouse synovial membrane, whereas safranin O-fast green staining was performed to detect cartilage injury, NLRP3 inflammasome and inflammatory factor levels in tissues. LF suppressed M1 polarization of macrophages, reduced M1 cell proportion and inflammatory factor levels, and suppressed the activation of NLRP3 inflammasome. After NLRP3 knockdown, LF did not further suppress the M1 polarization of macrophages. Pull-down assay suggested that LF bound to NLRP3. As revealed by mouse experimental results, LF inhibited bone injury in mice, decreased M1 cell infiltration and inflammatory response in tissues, and inhibited NLRP3 inflammasome expression in tissues. LF targets NLRP3 to suppress the M1 polarization of macrophages and decrease tissue inflammation in RA.

A rapid, high-throughput, and sensitive PEG-precipitation method for SARS-CoV-2 wastewater surveillance.

The effective application of wastewater surveillance is dependent on testing capacity and sensitivity to obtain high spatial resolution testing results for a timely targeted public health response. To achieve this purpose, the development of rapid, high-throughput, and sensitive virus concentration methods is urgently needed. Various protocols have been developed and implemented in wastewater surveillance networks so far, however, most of them lack the ability to scale up testing capacity or cannot achieve sufficient sensitivity for detecting SARS-CoV-2 RNA at low prevalence. In the present study, using positive raw wastewater in Hong Kong, a PEG precipitation-based three-step centrifugation method was developed, including low-speed centrifugation for large particles removal and the recovery of viral nucleic acid, and medium-speed centrifugation for the concentration of viral nucleic acid. This method could process over 100 samples by two persons per day to reach the process limit of detection (PLoD) of 3286 copies/L wastewater. Additionally, it was found that the testing capacity could be further increased by decreasing incubation and centrifugation time without significantly influencing the method sensitivity. The entire procedure uses ubiquitous reagents and instruments found in most laboratories to obtain robust testing results. This high-throughput, cost-effective, and sensitive tool will promote the establishment of nearly real-time wastewater surveillance networks for valuable public health information.

Synthesis and isolation of dinuclear N,C-chelate organoboron compounds bridged by neutral, anionic, and dianionic 4,4'-bipyridine via reductive coupling of pyridines.

The reaction of Bppy(Mes)2 (BN1; ppy = 2-phenylpyridine) and BCH2ppy(Mes)2 (BN3) with the reducing reagent KC8 resulted in C-C bond formation via intermolecular radical coupling to generate the 4,4'-bipyridyl ligand compounds BN2 and BN4. Adding 1 equivalent of KC8 to a THF solution of BN2 and BN4 generated the 4,4'-bipyridyl radical anions BN2K and BN4K. The dianion species BN2K2 and BN4K2 could be obtained by adding 2 equivalents of KC8 to the THF solution of BN2 and BN4. In the presence of 2,2,2-cryptand or 18-crown-6, the radical anion salt BN2K(crypt) and the dianion salt BN2K2(18c6)2 were isolated for single-crystal X-ray diffraction analysis. Structural, spectroscopic, and computational studies were performed on the three species of BN2 derivatives (neutral, radical anion, and dianion species). BN2 and BN4 were stable and did not undergo photoisomerization or photoelimination under UV light irradiation.

Anti-tumour necrosis factor-alpha agent therapy, compared with conventional therapy, reduces the relapse of uveitis in patients with behçet's disease: A systematic review of controlled trials.

Purpose: Anti-tumour necrosis factor-alpha (TNF-α) agents are often used for Behçet's disease (BD) in clinical practice, but they have not been validated by a high level of evidence. We systematically reviewed published controlled trials to investigate the efficacy and safety of anti-TNF-α therapy and summarize the efficacy of anti-TNF-α therapy relative to the available therapeutic options. Methods: A systematic database search was conducted (PubMed, Embase and Cochrane) using specific search terms. All controlled studies of anti-TNF-α treatment of BD patients prior to December 2021 were included. Single-arm studies were excluded. The decision of whether to incorporate data into the meta-analysis or summarize the data by qualitative synthesis was based on the results of the literature screening. Results: Of 4389 screened studies, 13 (total 778 patients) were included in accordance with our retrieval strategy, comprising 1 randomized controlled trial, 1 prospective study, 10 retrospective studies, and 1 multicentre open-label study. Ten studies (76.9%) involved Behçet's uveitis (BU), 1 involved intestinal BD, and the other studies had undefined subtypes. Subgroup reviews were conducted according to the control drug. Four studies involving 167 participants reported relapse rates. Meta-analysis of three of these studies demonstrated that, compared with traditional immunosuppressant (TIS) therapy, anti-TNF-α therapy reduced the relapse rates in patients with BU. In targeted drug comparison studies, the efficacy appeared to be similar between the anti-TNF-α agent and interferon in BU patients. The rates of adverse events were comparable between a variety of different therapeutic controls. Serious adverse events were not observed in 53.8% (7/13) of the studies. Conclusions: Compared with TIS therapy, anti-TNF-a therapy reduces the relapse of uveitis in patients with BD. However, the evidence regarding anti-TNF-α therapy is very limited for the full spectrum of BD subtypes, which calls for caution.

CO, CO2 and CS2 activation by divalent ytterbium hydrido complexes.

Treatment of a divalent ytterbium hydride complex [(TpAd,iPr)Yb(H)(THF)] (TpAd,iPr = hydrotris(3-adamantyl-5-isopropyl-pyrazolyl)borate) (1) with CO, CO2 and CS2 resulted in the formation of a divalent ytterbium ethenediolate complex [(TpAd,iPr)Yb]2(cis-OCHCHO) (2), a formate complex [(TpAd,iPr)Yb(κ2-O2CH)(THF)] (3), and a trivalent ytterbium ethenetetrathiolate complex [(TpAd,iPr)YbIII]2(C2S4) (4), respectively. DFT calculations were carried out to elucidate the reaction profiles of complexes 3 and 4.

Cytopathological characteristics of angioimmunoblastic T-cell lymphoma diagnosed by fine needle aspiration.

OBJECTIVE: To analyse the cytopathological features of fine needle aspiration (FNA) in angioimmunoblastic T-cell lymphoma (AITL) diagnostics. METHODS: Fine needle aspiration lymph node biopsy samples from 12 patients with AITL were collected at a single centre between January 2014 and December 2020. The clinical, cytological and histopathological characteristics were retrospectively analysed. RESULTS: Three male and six female patients with AITL who had a median onset age of 65 years (range 51-74 years) and a mean follow-up period of 29 months (range 12-47 months) were included. The FNA cytological and morphological analysis of the reactive lymph node background revealed diffusely distributed non-homogeneous mixed lymphocytes, including mature small lymphocytes, medium-sized lymphoid cells, immune cells, and plasma cells; some mixed eosinophils, macrophages, and an occasional mixture of visible and medium-sized lymphocytes and epithelioid cells were observed. Mitotically active lymphocytes and sporadic pigmented bodies were observed occasionally. An abnormal proliferation of follicular dendritic cells observed under the microscope is important for AITL diagnosis, and these cells are often distributed in a scattered pattern of small clusters with many nuclear morphologies. Branched capillaries are another important diagnostic clue. Two patients with AITL who achieved clinical remission after treatment experienced recurrence and were diagnosed using FNA and cell block immunohistochemistry. CONCLUSIONS: Fine needle aspiration provides clues for the diagnosis of AITL in special clinical situations, and cell block immunohistochemistry is worthy of further exploration.

Synthesis of pyrimidines from dinitrogen and carbon.

The element nitrogen and nitrogenous compounds are vital to life. The synthesis of nitrogen-containing compounds using dinitrogen as the nitrogen source, not through ammonia, is of great interest and great value but remains a grand challenge. Herein, we describe a strategy to realize this transformation by combining the heterogeneous approach with the homogeneous methodology. The N2 molecule was first fixed with carbon and LiH through a one-pot heterogeneous process, forming Li2CN2 as an 'activated' nitrogen source with high efficiency. Then subsequent homogeneous treatments of Li2CN2 to construct the organic synthon carbodiimide and the RNA/DNA building block pyrimidines were fulfilled. By using 15N2 as the feedstock, their corresponding 15N-labeled carbodiimide and pyrimidines were readily obtained. This homogeneous-heterogeneous synergy strategy will open a new chapter for N2 transformation.

A mononuclear divalent ytterbium hydrido complex supported by a super-bulky scorpionate ligand.

The first mononuclear divalent ytterbium hydride complex [(TpAd,iPr)Yb(H)(THF)] (TpAd,iPr = hydrotris(3-adamantyl-5-isopropyl-pyrazolyl)borate) (2) bearing a terminal hydrido ligand was obtained by hydrogenolysis of the benzyl precursor in hexane. Complex 2 exhibited two different reaction patterns towards allenes: Yb-H addition with cyclohexylallene and deprotonation of 1,1-dimethylallene.

Scandium and lanthanum hydride complexes stabilized by super-bulky penta-arylcyclopentadienyl ligands.

Hydrogenolysis of the half-sandwich penta-arylcycopentadienyl-supported rare-earth metal dibenzyl complexes [(CpAr5)Ln(p-CH2-C6H4-Me)2(THF)] (CpAr5 = C5Ar5, Ar = 3,5-iPr2-C6H3; Ln = Sc, La) afforded a bimetallic scandium complex [(CpAr5)Sc(H)(µ-OC4H9)]2 (2) with two terminal hydrido ligands, and a double-sandwich bimetallic lanthanum hydride complex [(CpAr5)La(µ-H)]2 (4) bearing the reduced CpAr5 ligand. DFT calculations were conducted to elucidate the reaction profiles.

Trinuclear scandium methylidyne complexes stabilized by pentamethylcyclopentadienyl ligands.

The first examples of scandium methylidyne complexes [(Cp*)Sc(µ2-X)]3(µ3-CH) (Cp* = C5Me5; X = Br, Me, OMe), free of Lewis acids, can be achieved in high yields from [(Cp*)ScMe2]2 through a facile route. The chemical and geometrical flexibility to incorporate organic substrates indicates a rich chemistry of complex [(Cp*)Sc(µ2-OMe)]3(µ3-CH).

Synergistic Effects of Graphene and Ammonium Polyphosphate Modified with Vinyltrimethoxysilane on the Properties of High-Impact Polystyrene Composites.

Ammonium polyphosphate (APP) was modified with a silane coupling agent (vinyltrimethoxysilane, Si-171), and then the synergistic flame retarding effect of graphene and surface-modified APP (APP@Si-171) on high-impact polystyrene (HIPS) was investigated. Surface modification and thermal stability characterization of APP were analyzed by Fourier transform infrared spectroscopy (FTIR), energy dispersive spectrometer (EDS), scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). The results showed that surface-modified APP (APP@Si-171) exhibited significantly better dispersion and less agglomeration tendencies compared with pure APP. A series of target HIPS composites containing different mass fractions of the two flame retardants were prepared by melt blending. TGA and cone calorimeter tests (CCT) were conducted to quantitatively investigate the thermal and flammability properties of the composites, respectively. Results from TGA and CCT demonstrated that the addition of the flame retardants delayed the onset and peak temperatures in differential thermogravimetry (DTG) curves and weakened the peak heat release rate (PHRR) and total heat release (THR). Moreover, the synergistic effect index (SE) was employed to quantify the synergistic behavior between the two fillers, and the results showed that APP@Si-171 and graphene had a synergistic effect on improving the thermal stability and flame retardancy of HIPS.

Selective homo- and cross-desilacoupling of aryl and benzyl primary silanes catalyzed by a barium complex.

Under mild conditions (25 °C, 5 mol% cat.), highly selective homo- and cross-desilacoupling of aryl and benzyl primary silanes to secondary silanes was achieved by the use of the heteroleptic barium aminobenzyl complex [(TpAd,iPr)Ba(CH2C6H4NMe2-o)] (TpAd,iPr = hydrotris(3-adamantyl-5-isopropyl-pyrazolyl)borate) (1) as a catalyst. Dihydrosilanes originating from catalytic redistribution and cross-desilacoupling reactions were isolated in fine yields, which demonstrates the feasible application of the barium complex in the syntheses of secondary aryl- and benzylsilanes.

Mononuclear calcium complex as effective catalyst for alkenes hydrogenation.

Hydrogenolysis of the scorpionate-supported calcium benzyl complex [(TpAd,iPr)Ca(p-CH2C6H4-Me)(THP)] (TpAd,iPr = hydrotris(3-adamantyl-5-isopropyl-pyrazolyl)borate, THP = tetrahydropyran) (2-THP) afforded the mononuclear calcium hydrido complex [(TpAd,iPr)Ca(H)(THP)] (3). Under mild conditions (40 °C, 10 atm H2, 5 mol% cat.), complex 3 effectively catalyzed the hydrogenation of a variety of alkenes, including activated alkenes, semi-activated alkenes, non-activated terminal and internal alkenes. Mononuclear calcium unsubstituted alkyl complex [(TpAd,iPr)Ca{(CH2)4Ph}(THP)] (6), proposed as the catalytic hydrogenation intermediate, was isolated and structurally characterized.

Barium tetraalkylaluminate complexes supported by the super-bulky hydrotris(pyrazolyl)borate ligand.

A heteroleptic barium aminobenzyl complex [(TpAd,iPr)Ba(o-CH2C6H4-NMe2)] (1) was obtained in excellent yield from a simple one-pot reaction. Treatment of [(TpAd,iPr)Ba(o-CH2C6H4-NMe2)] (1) with two equivalents of AlR3 (R = Me, Et) led to the formation of barium tetraalkylaluminate complexes [(TpAd,iPr)Ba(AlR4)]n (R = Me, n = 2, 2; R = Et, n = 1, 3) as dimers or monomers in the solid state. The TpAd,iPr ligand-free peralkylated barium complex [Ba(AlEt4)2]n was isolated by the addition of ten equivalents of AlEt3 under the same conditions. The donor-induced aluminate cleavage is not applicable when donor solvents are added to complexes 2 and 3. In the solution of complexes 2 and 3, the alkylaluminate moieties and TpAd,iPr ligands show a rapid fluxional behavior in [D8]toluene solution over the temperature range of -70 to 25 °C, without any significant decoalescence of the corresponding proton signals.

Monomeric thorium dihydrido complexes: versatile precursors to actinide metallacycles.

The monomeric actinide dihydrido complex [(CpAr*)(Cp*)ThH2(THF)] (2) bearing the super-bulky penta-arylcyclopentadienyl (CpAr* = C5Ar*5, Ar* = 3,5-tBu2-C6H3) and pentamethylcyclopentadienyl (Cp* = C5Me5) ligands was obtained for the first time. Complex 2 underwent unique Th-H addition reactions with various unsaturated compounds to afford the corresponding five-membered metallacycles, including the first example of actinide metallacyclopentyne [(CpAr*)(Cp*)Th(PhCH-C[triple bond, length as m-dash]C-CHPh)] (4).

Reversible addition and hydrogenation of 1,1-diphenylethylene with a barium complex.

Treatment of the barium hydride complex [(TpAd,iPr)Ba(µ-H)]2 (TpAd,iPr = hydrotris(3-adamantyl-5-isopropyl-pyrazolyl)borate) (1) with an excess amount of 1,1-diphenylethylene (DPE) afforded the corresponding mono-DPE adduct [(TpAd,iPr)Ba(η6-Ph)C(Ph)Me] (2) and di-DPE adduct [(TpAd,iPr)Ba(η6-Ph)C(Ph)-CH2-C(Ph)2Me] (3), which could convert to each other through a reversible equilibrium reaction. Complex 1 catalysed DPE hydrogenation under mild conditions (30 °C, 6 atm, 5 mol% cat.).

The etiology of acute meningitis and encephalitis syndromes in a sentinel pediatric hospital, Shenzhen, China.

BACKGROUND: Acute meningitis and encephalitis syndromes (AMES) is a severe neurological infection which causes high case fatality and severe sequelae in children. To determine the etiology of childhood AMES in Shenzhen, a hospital-based study was undertaken. METHODS: A total of 240 cerebrospinal fluid (CSF) samples from 171 children meeting the case definition were included and screened for 12 common causative organisms. The clinical data and conventional testing results were collected and analyzed. Whole genome sequencing was performed on a Neisseria meningitidis isolate. RESULTS: A pathogen was found in 85 (49.7%) cases; Group B Streptococcus (GBS) was detected in 17 cases, Escherichia coli in 15, Streptococcus pneumoniae in 14, enterovirus (EV) in 13, herpes simplex virus (HSV) in 3, N. meningitidis in 1, Haemophilus influenzae in 1, and others in 23. Notably, HSV was found after 43 days of treatment. Twelve GBS and 6 E. coli meningitis were found in neonates aged less than 1 month; 13 pneumococcal meningitis in children aged > 3 months; and 12 EV infections in children aged > 1 year old. The multilocus sequence typing of serogroup B N. meningitidis isolate was ST-3200/CC4821. High resistance rate to tetracycline (75%), penicillin (75%), and trimethoprim/sulfamethoxazole (75%) was found in 4 of S. pneumoniae isolates; clindamycin (100%) and tetracycline (100%) in 9 of GBS; and ampicillin (75%) and trimethoprim/sulfamethoxazole (67%) in 12 of E. coli. CONCLUSIONS: The prevalence of N. meningitidis and JEV was very low and the cases of childhood AMES were mainly caused by other pathogens. GBS and E. coli were the main causative organisms in neonates, while S. pneumoniae and EV were mainly found in older children. HSV could be persistently found in the CSF samples despite of the treatment. A better prevention strategy for GBS, the introduction of pneumococcal vaccine, and incorporation of PCR methods were recommended.