SARS-CoV-2 Gastrointestinal Shedding in Hospitalized Children

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a respiratory virus that can cause gastrointestinal (GI) symptoms, with studies demonstrating detection of stool viral RNA weeks after respiratory tract clearance. It is unknown if children who test negative for SARS-CoV-2 on a nasopharyngeal (NP) swab may be shedding the virus in their stool. OBJECTIVE: To measure the prevalence of SARS-CoV-2 stool shedding in children with positive and negative SARS-CoV-2 NP polymerase chain reactions (PCR) tests, and to determine clinical factors associated with GI shedding. METHODS: In this cross-sectional study, we enrolled hospitalized patients 0 to 21 years old with a positive or a negative SARS-CoV-2 NP PCR test who had respiratory and/or GI symptoms. Participants were surveyed, and stool samples were sent for viral PCR testing. Fisher’s exact test was used to evaluate bivariate associations of stool PCR test positivity with categorical variables. RESULTS: Sixty-seven patients were consented; 34 patients did not provide stool samples so 33 patients were included: 17 NP-positive and 16 NP-negative for SARS-CoV-2. Eight of the 17 NP-positive patients had a positive stool PCR test for SARS-CoV-2, while none of the 16 SARS-CoV-2 NP-negative patients had a positive result (P < .01). For the 17 SARS-CoV-2 NP-positive patients, GI symptoms were associated with a positive stool PCR test (P = .05) for SARS-CoV-2, but this association was not found for all 33 patients (P = .11). No associations were found with patients in an immunocompromised state or those with a comorbid condition, fever and/or chills, respiratory symptoms, headache and/or myalgias, or anosmia and/or ageusia. CONCLUSIONS: SARS-CoV-2 GI shedding is common and associated with GI symptoms in NP-positive children, with 47% having positive stool PCRs for SARS-CoV-2. GI shedding was not demonstrated in SARS-CoV-2 NP-negative children.

Endometrial injury concurrent with hysteroscopy increases the expression of Leukaemia inhibitory factor: a preliminary study

Objective It is not known by which mechanism endometrial injury increases pregnancy rates. Leukaemia inhibitory factor (LIF) is a cytokine involved in wound healing and implantation. The aim of this study was to determine the change in endometrial LIF mRNA expression before and after mechanical injury during hysteroscopy. Methods Forty patients with a history of two or more unsuccessful implantations who decided to undergo hysteroscopy in the proliferative phase were divided into two equal groups: one with endometrial injury (scratching group) and the other with noninjury (control group). Endometrial sampling was conducted before injury on the patients in the scratching group, and then injury was performed with monopolar needle forceps. Only diagnostic hysteroscopy was performed on the patients in the control group. Endometrial tissues were collected using a Pipelle catheter between Days 20 and 23 of the mid-luteal phase of the next cycles in both the scratching and control groups. Endometrial LIF mRNA expression was evaluated with the use of reverse-transcription polymerase chain reactions. Results Relative changes in mRNA expression levels of the LIF gene in endometrial samples taken before and after injury were calculated using the 2-ΔΔCt method, and the fold changes obtained were compared between and within the groups. Compared with preinjury values, an 11.1-fold increase was found in postinjury LIF mRNA expression in patients with monopolar forceps injury (p < 0.001). There was a 3.9-fold significant increase in postinjury LIF mRNA levels compared with those in the control group (p < 0.02). Conclusions The fertility-promoting effect of hysteroscopy-guided mechanical endometrial injury may be mediated by LIF mRNA.

Yersinia massiliensis (Enterobacteriales: Enterobacteriaceae) in the host Anaphes nitens (Hymenoptera: Mymaridae): first report of association with insects

Endosymbiont bacteria can affect biological parameters and reduce the effectiveness of natural enemies in controlling the target insect. The objective of this work was to identify endosymbiont bacteria in Anaphes nitens (Girault, 1928) (Hymenoptera: Mymaridae), the main natural enemy used to manage Gonipterus platensis (Marelli, 1926) (Coleoptera: Curculionidae). Genomic DNA from six A. nitens populations was extracted and polymerase chain reactions (PCR) were performed with the primers to detect endosymbiont bacteria in this insect. The PCR products were amplified, sequenced, and compared with sequences deposited in the GenBank for the bacteria identification. All A. nitens populations had the bacterium Yersinia massiliensis (Enterobacteriales: Enterobacteriaceae). This bacterium was originally described as free-living, and it is associated with and composes part of the A. nitens microbiota. This is the first report of Y. massiliensis in an insect host.

Anti-Inflammatory Activities of the Ethanol Extract of Prasiola japonica, an Edible Freshwater Green Algae, and Its Various Solvent Fractions in LPS-Induced Macrophages and Carrageenan-Induced Paw Edema via the AP-1 Pathway

Prasiola japonica possesses several biological activities. However, reports on the anti-inflammatory activities and molecular mechanisms of its different solvent fractions remain limited. In this study, we investigated the potential anti-inflammatory activities of P. japonica ethanol extract (Pj-EE) and four solvent fractions of Pj-EE made with hexane (Pj-EE-HF), chloroform (Pj-EE-CF), butanol (Pj-EE-BF), or water (Pj-EE-WF) in both in vitro (LPS-induced macrophage-like RAW264.7 cells) and in vivo (carrageenan-induced acute paw edema mouse models) experiments. The most active solvent fraction was selected for further analysis. Various in vitro and in vivo assessments, including nitric oxide (NO), cytokines, luciferase assays, real-time polymerase chain reactions, and immunoblotting analyses were performed to evaluate the underlying mechanisms. In addition, the phytochemical constituents were characterized by Liquid chromatography-tandem mass spectrometry. In in vitro studies, the highest inhibition of NO production was observed in Pj-EE-CF. Further examination revealed that Pj-EE-CF decreased the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells and suppressed subsequent AP-1-luciferase activity by inhibition of phosphorylation events in the AP-1 signaling pathway. Pj-EE-CF treatment also demonstrated the strongest reduction in thickness and volume of carrageenan-induced paw edema, while Pj-EE-BF showed the lowest activity. Furthermore, Pj-EE-CF also reduced gene expression and cytokines production in tissue lysates of carrageenan-induced paw edema. These findings support and validate the evidence that Pj-EE, and especially Pj-EE-CF, could be a good natural source for an anti-inflammatory agent that targets the AP1 pathway.

Conjunctival polymerase chain reactions in COVID-19 patients and its correlation with clinical and paraclinical indexes

Background and Objectives: This study aimed to detect SARS-CoV-2 in conjunctival samples of COVID-19 patients to investigate the transmission route of COVID-19 and its correlation with laboratory indexes. Materials and Methods: In this cross-sectional study, 44 COVID-19 patients were tested for conjunctival PCR in Ayatollah Rouhani hospital of Babol, Iran, in January and February 2021. The conjunctival samples were collected using a conjunctival swab and suspended in a viral transport medium. After RNA extraction and cDNA synthesis, real-time PCR was performed to investigate the SARS-CoV-2 genome in samples. The ocular manifestations and laboratory indexes were evaluated for all patients. Results: Among 44 COVID-19 patients, 6 samples (13.63%) were positive in terms of conjunctival PCR. The mean ± SD age of conjunctival PCR-positive patients was 76.17 ± 16.61-year-old, while conjunctival PCR-negative COVID-19 pa- tients were aged 57.54 ± 13.61-year-old (p <0.05). D-dimer serum level is significantly higher in conjunctival PCR-positive COVID-19 patients (4001.00 ± 3043.36 µg/ml) compared to normal individuals (496.80 ± 805.92 µg/ml, p <0.01). Conclusion: Our study showed that the conjunctiva and tear contain the SARS-CoV-2 in COVID-19 patients as a possible transmission route.

Effect of Xuezhikang Therapy on Expression of Pulmonary Hypertension Related miR-638 in Patients With Low HDL-C Levels

Objective: Low plasma level of high-density lipoprotein cholesterol (HDL-C) associated with poor outcomes in several cardiovascular diseases, including pulmonary arterial hypertension (PAH). Regulation of miR-638 have been proved to be associated with PAH. The aim of this study was to evaluate the expression of miR-638 after Xuezhikang (XZK) therapy in patients with low HDL-C.Methods: Plasma levels of miR-638 were quantified by real-time polymerase chain reactions in 20 patients with PAH and 30 healthy controls. A total of 40 subjects with low HDL-C were assigned to receive an XZK therapy for 6 months. The miR-638 expression profiles were detected in PAH patients, XZK-treated subjects and lovastatin treated pulmonary arterial smooth muscle cells (PA-SMCs).Results: The relative expression level of miR-638 in the plasma was lower in the PAH patients than that in the controls (p &lt; 0.001). An increase of 11.2% from baseline in the HDL-C level was found after XZK therapy (p &lt; 0.001). The relative expression of miR-638 was increased after XZK treatment (p &lt; 0.01). The changes of miR-638 were inversely associated with baseline HDL-C levels. A significantly reduction in miR-638 expression were found in PDGF-BB-treated hPA-SMCs compared to the control cells, and the pre-treatment of the cells with lovastatin significantly re-gain the expression levels in miR-638.Conclusion: In patients with low HDL-C levels, XZK therapy raised the expression of miR-638, suggesting that the potential therapeutic effect of XZK in PAH patients with low serum HDL-C levels deserves further exploration.

RHD Genotypes in a Chinese Cohort of Pregnant Women

RHD variants in D¯ Chinese pregnant women arose difficulties in management during pregnancy. Therefore, this study aims to precisely manage D¯ pregnant women by evaluating the spectrum of RHD mutations in D¯ pregnant women and getting insight into the possible rare alleles of RHD. A total of 76 D¯ pregnant women were analyzed by performing polymerase chain reactions with sequence-specific primers (PCR-SSP), the 10 RHD exons Sanger sequencing, RHD zygosity detection, and mRNA sequencing (mRNA-seq). About 40% of alleles are variations of RHD, including RHD 1227A homozygous, RHD-CE(2-9)-D, et al. Therefore, we developed a molecular diagnostic strategy for Chinese women, and most D¯ pregnant women can be diagnosed with this simple decision tree. After RHD genotyping for D¯ pregnancy women, we eliminated at least 15% unnecessary ante- and postpartum injections of Rh immunoglobulin (RhIG). As the first pedigree study and the first functional analysis under physiological conditions, mRNA-seq revealed that c.336-1G&gt;A mutation mainly led to the inclusion of the intron 2, which indirectly explained the D¯ phenotype in this family. We also developed a robust protocol for determining fetal RhD status from maternal plasma. All 31 fetuses were predicted as RhD positive and confirmed the RhD status after birth.

Molecular characterization of pathogenic OTOA gene conversions in hearing loss patients

Bi-allelic loss-of-function variants of OTOA are a well-known cause of moderate-to-severe hearing loss. Whereas non-allelic homologous recombination-mediated deletions of the gene are well known, gene conversions to pseudogene OTOAP1 have been reported in the literature but never fully described nor their pathogenicity assessed. Here, we report two unrelated patients with moderate hearing-loss, who were compound heterozygotes for a converted allele and a deletion of OTOA. The conversions were initially detected through sequencing depths anomalies at the OTOA locus after exome sequencing, then confirmed with long range polymerase chain reactions. Both conversions lead to loss-of-function by introducing a premature stop codon in exon 22 (p.Glu787*). Using genomic alignments and long read nanopore sequencing, we found that the two probands carry stretches of converted DNA of widely different lengths (at least 9 kbp and around 900 bp, respectively).

Microfluidic Chain Reaction: Conditional, Structurally Programmed Propagation of Capillary Flow Events

Chain reactions are characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (e.g. combustion engines), nuclear and biotechnological (e.g. polymerase chain reaction) applications.1-5 At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling domino and Rube Goldberg machines. Appositely, the microfluidic lab-on-a-chips (also called a micro total analysis system),6,7 was envisioned as an integrated chip, akin to microelectronic integrated circuits, yet in practice remain dependent on cumbersome peripherals, connections, and a computer for automation.8-11 Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible.12-19 Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are 3D printed, and powered by the free-energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (i) the sequential release of 300 aliquots across chained, interconnected chips, (ii) a protocol for SARS-CoV-2 antibodies detection in saliva, and (iii) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ, and can form frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations

Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.