Opportunistic pathogens increased and probiotics or short-chain fatty acid-producing bacteria decreased in the intestinal microbiota of pneumonia inpatients during SARS-CoV-2 Omicron variant epidemic.

The global pandemic of COVID-19 has been over four years, and the role of intestinal microbiota in the occurrence and development of COVID-19 needs to be further clarified. During the outbreak of SARS-CoV-2 Omicron variant in China, we analyzed the intestinal microbiome in fecal samples from inpatients with pneumonia and normal individuals in January 2023. The microbiota composition, alpha diversity, beta diversity, differential microbial community, co-occurrence networks, and functional abundance were analyzed. The results showed significant differences in microbiota composition between the two groups. In pneumonia group, the abundance of Bifidobacterium, Blautia, Clostridium, and Coprococcus decreased, while the abundance of Enterococcus, Lactobacillus, and Megamonas increased. Through LEfSe analysis, 37 marker microbiota were identified in pneumonia group. Co-occurrence network analysis found that Lachnospiraceae was critical for the interaction of intestinal microbiota, and the anti-inflammatory bacteria Blautia was negatively correlated with the pro-inflammatory bacteria Ruminococcus. Functional prediction found the up-regulation of steroid biosynthesis, geraniol degradation, and mRNA surveillance pathway in pneumonia group. In conclusion, opportunistic pathogens increased and probiotics, or short-chain fatty acid-producing bacteria, decreased in the intestinal microbiota of pneumonia inpatients during the Omicron epidemic. Blautia could be used as a probiotic in the treatment of pneumonia patients in the future.

Gas-Responsive and Self-Powered Visual Composite Langmuir-Blodgett Films for Ultrathin Gas Sensors.

The complex and variable environments are challenging the development of related detection and analysis. Ammonia (NH3) and hydrogen chloride (HCl) gases are both commonly used in industry, but they are considered to be toxic and corrosive substances that can threaten human health and the environment. Therefore, it is necessary here to develop a convenient, sensitive, and reliable sensor device for acid-alkali gas detection. Herein, we propose the synthesis strategy of an ultrathin film gas sensor based on the pH-responsive, self-powered, and visible composite Langmuir-Blodgett (LB) films. In our work, the LB films with nanometric thicknesses are obtained based on the sensitive materials of two novel carbazole structural sensitizers (abbreviated as CS-35 and CS-37) and several dye molecules. The composite LB films are formed with Carbazole samples and dye molecules through hydrogen bonding, π-π stacking, synergistic electrostatic interactions, and hydrophobic interactions, existing as J-aggregate or H-aggregate. The formation of high-quality and uniform Langmuir films is confirmed with transmission electron microscope (TEM), UV-vis spectrum, atomic force microscopy (AFM), and other measurements. In addition, based on the simple protonation and deprotonation, the prepared LB films can be assembled into a visual sensor for the response of pH gases. The response is confirmed by the study of ultraviolet spectroscopy and electrical output in vertical contact separation mode, which potentially unlocks a sustainable future for the application of ultrathin self-powered gas sensors.

A novel tonicity-responsive microRNA miR-23a-5p modulates renal cell survival under osmotic stress through targeting heat shock protein 70 HSPA1B.

There is growing evidence that microRNAs (miRNAs) are implicated in cellular adaptation to osmotic stress, but the underlying osmosignaling pathways are still not completely understood. In this study, we found that a passenger strand miRNA, miR-23a-5p, was significantly downregulated in response to high NaCl treatment in mouse inner medullary collecting duct cells (mIMCD3) through an miRNA profiling assay. The decrease of miR-23a-5p is hypertonicity-dependent and osmotolerant cell type-specific. Knockdown of miR-23a-5p increased cellular survival and proliferation in mIMCD3. In contrast, miR-23a-5p overexpression repressed cell viability and proliferation under hypertonic stress. RNA deep-sequencing revealed that a heat shock protein 70 (HSP70) isoform, HSP70 member 1B (HSPA1B), was significantly increased under hypertonic treatment. Based on the prediction analysis by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and TargetScan, and a further validation via a dual-luciferase assay, HSPA1B was identified as a potential target of miR-23a-5p. Overexpressed miR-23a-5p suppressed HSPA1B, whereas downregulated miR-23a-5p promoted HSPA1B expression in mIMCD3. In addition, an in vivo study demonstrated that there is a reverse correlation between the levels of miR-23a-5p and HSPA1B in mouse renal inner medulla (papilla) that is exposed to extremely high osmolality. In summary, this study elucidates that passenger strand miR-23a-5p is a novel tonicity-responsive miRNA. The downregulation of miR-23a-5p facilitates cellular adaptation to hypertonic stress in mammalian renal cells through modulating HSPA1B.

Hydroxypropyl methyl cellulose reinforced conducting polymer hydrogels with ultra-stretchability and low hysteresis as highly sensitive strain sensors for wearable health monitoring.

Conducting polymer hydrogels have emerged as promising materials to fabricate highly sensitive strain sensors. However, due to weak bindings between conducting polymer and gel network, they usually suffer from limited stretchability and large hysteresis, failing to achieve wide-range strain sensing. Herein, we combine hydroxypropyl methyl cellulose (HPMC), poly (3,4-ethylenedioxythiophene):poly (styrene sulfonic acid) (PEDOT: PSS) with chemically cross-linked polyacrylamide (PAM) to prepare a conducting polymer hydrogel for strain sensors. Owing to abundant hydrogen bonds between HPMC, PEDOT:PSS and PAM chains, this conducting polymer hydrogel exhibits high tensile strength (166 kPa), ultra-stretchability (>1600 %) and low hysteresis (<10 % at 1000 % cyclic tensile strain). The resultant hydrogel strain sensor shows ultra-high sensitivity, wide strain sensing ranges of 2-1600 %, and excellent durability and reproducibility. Finally, this strain sensor can be used as wearable sensor to monitor vigorous human movement and fine physiological activity, and services as bioelectrodes for electrocardiograph and electromyography monitoring. This work provides new horizons to design conducting polymer hydrogels for advanced sensing devices.

Ionic conductive hydroxypropyl methyl cellulose reinforced hydrogels with extreme stretchability, self-adhesion and anti-freezing ability for highly sensitive skin-like sensors.

Ionically-conductive hydrogels are attracting increasing interest as skin-like sensors, however, the fabrication of ion-conductive hydrogels with excellent mechanical properties, high conductivity, self-adhesion and anti-freezing ability for high-performance sensors remains a challenge. Herein, a highly ion-conductive hydrogel is prepared by introducing LiCl into polyacrylamide/hydroxypropyl methyl cellulose (PAM/HPMC) composite hydrogel. The introduction of LiCl simultaneously endows the PAM/HPMC/LiCl hydrogel with outstanding stretchability (1453 %), high tensile strength (135 kPa), skin-like elasticity (9.18 kPa), high conductivity (7.85 S/m), good adhesiveness and wide operating temperature range. Impressively, this ion-conductive hydrogel can be utilized in skin-like sensor, which achieves high strain sensitivity (GF = 11.19) with wide sensing ranges (up to 600 %), and excellent endurance over 250 consecutive stretching. As a result, the wearable sensor assembled from the hydrogels can be used to detect complex human activities with high stability even at -40 °C. This work promotes the development of ion-conductive hydrogels with broad operating temperature in advanced sensory platform.

Interacting domains of P14-3-3 and actin involved in protein-protein interactions of living cells.

14-3-3 proteins are conserved regulatory proteins present in all eukaryotic cells that control numerous cellular activities via targeted protein interactions. To elucidate the interaction between P14-3-3 from Physarum polycephalum and actin in living cells, PCR and DNA recombination were used to generate various P14-3-3 and actin constructs. Yeast two-hybrid assay and FRET were employed to characterize the interaction between P14-3-3 and actin. The two-hybrid assay indicated that P14-3-3 N-terminal 76-108 amino acids and the C-terminal 207-216 amino acids played an important role in mediating interactions with actin, and the actin N-terminal 1-54 amino acids and the C-terminal 326-376 amino acids are also crucial in the interactions with the mPa, a P14-3-3 with mutations at Ser62 (Ser62 â†’ Gly62). Mutations to potential phosphorylation sites did not affect interactions between P14-3-3 and actin. FRET results demonstrated that P14-3-3 co-localized with actin with a FRET efficiency of 22.2% and a distance of 7.4 nm and that P14-3-3 N-terminal 76-108 and C-terminal 207-216 amino acids were important in mediating this interaction, the truncated actin peptides without either the N-terminal 1-54 or C-terminal 326-376 amino acids interacted with P14-3-3, consistent with the results obtained from the yeast two-hybrid assay. Based on data obtained, we identified critical actin and P14-3-3 contact regions.

Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites.

The roles of 14-3-3 proteins in the lower eukaryotes are still elusive. We isolated a cDNA encoding the 14-3-3 protein (P14-3-3) from the lower eukaryote Physarum polycephalum. This P14-3-3 gene was then inserted downstream of the Gal4 DNA-binding domain in the yeast expression vector pGBKT7. The recombinant vector was transformed into auxotrophic AH109 and Y187 yeast cells to detect the activation of Gal4-regulated gene expression mediated by P14-3-3. The results showed that three reporter genes (ADE2, HIS3, and lacZ) could be normally expressed, indicating that the transcriptional activation function of P14-3-3 was retained. We subsequently used a truncated P14-3-3 peptides and mutant peptides to study the activation of the Gal4-regulated genes ADE2, HIS3, and lacZ. We found that deletion of the N-terminal second dimer-binding motif (DBM2) sequence or the C-terminal coil sequence led to the loss of P14-3-3's transcriptional activation function. Specifically, any mutation at the potential phosphorylation sites (Ser62 and Ser67) on DBM2 or at the C-terminal potential phosphorylation site (235ThrSer236) led to the loss of the transcriptional activation function of P14-3-3. Taken together, these observations suggest that the transcriptional activation function of P14-3-3 in lower eukaryotes is related to DBM2 and the C-terminal coil structures.

Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum.

BACKGROUND: Serine/arginine (SR) protein-specific kinases (SRPKs) are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS) of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379) from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. RESULTS: We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS). Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328) was localized in the alkaline Omega-loop of a helix-loop-helix motif (HLHM) of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. CONCLUSION: This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Omega-loop structure could play a crucial role in the NLS function of PSRPK.

A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein, ASF/SF2.

A 1591-bp cDNA of a serine-rich protein kinase (SRPK)-like protein has been identified in Physarum polycephalum (GenBank accession No. DQ140379). The cDNA contains two repeat sequences at bp 1-153 and bp 395-547. The encoding sequence is 56% homologous to human SRPK1 and is named Physarum SRPK (PSRPK). Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains (CDs). However, divergent motifs between the N-terminal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphorylation assay to investigate the phosphorylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coli cells. Autoradiographic examination showed that both recombinant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could phosphorylate ASF/SF2 and a truncated form of ASF/SF2 that contains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and function of PSRPK demonstrates that it is a new member of the SRPK family.

Effect of oxidative stress from nanoscale TiO2 particles on a Physarum polycephalum macroplasmodium under dark conditions.

Information regarding the effect of nanoscale titanium dioxide particles (nTiO2) on the environment under dark conditions is scarce, and the effect of nTiO2 on fungi is largely unknown. Due to its huge size and high sensitivity to external stimuli, the slime mold fungi cell, Physarum polycephalum macroplasmodium, was utilized as a novel subject for the toxicity investigations in the present study, and oxidative stress from nTiO2 on the macroplasmodium was assessed under dark conditions. Short exposure (2-3 h) caused an intracellular reactive oxygen species (ROS) imbalance, and an anti-oxidative mechanism was activated from intermediate doses of nTiO2 (5-18 mg/mL). At long exposure times (~3 days), relatively low doses of nTiO2 (≤9 mg/mL) stimulated the growth of macroplasmodium and oxidative stress without DNA damage, whereas higher doses of nTiO2 (≥15 mg/mL) led to growth inhibition, significant DNA oxidative damage, and activation of the DNA single-strand repairing system. Although DNA oxidative damage was decreased to the same level as the control group by the supplementation of the anti-oxidant vitamin C, growth of the macroplasmodium failed to be completely restored. We inferred that nTiO2 induced a complicated toxicity effect on P. polycephalum in addition to DNA oxidative damage. Taken as a whole, the present study implied the probability of using P. polycephalum macroplasmodium for toxicity studies at the single-cell level, indicating that nTiO2 could induce oxidative stress or damage in P. polycephalum even under dark conditions and suggesting that the release of nTiO2 could lead to a growth imbalance of slime molds in the environment.

[The characteristics of PSCL32.5 protein from Physarum polycephalum and the variation of the content through cell cycle].

Crude fraction of Arg/Ser-rich proteins (SR proteins) were isolated from plasmodia of Physarum polycephalum and immunoassayed by western blot with a monoclonal antibody against SC35 protein from HeLa cell. Two polypeptides were detected by the antibody, suggesting that they were SCL(SC35-like) proteins. The SCL proteins have their mass weight of 32.5 kD and 82.5 kD, respectively, and so were termed PSCL32.5 and PSCL82.5 in this paper. The PI of PSCI32.5 was ascertained as 6.19 by IEF after a further purification of the protein with SDS-PAGE. The densitometric scanning of the western blot bands of PSCI32.5 isolated at different phases of cell cycle of P. polycephalum demonstrated that the relative content of the protein varied through the cell cycle: it appeared as the lowest at early S phase, showed increases from S phase to G2 phase, and peaked at late G2 phase.

[Isolation and identification of SARS-coronavirus in nasal and throat swabs collected from clinically diagnosed SARS patients].

OBJECTIVE: To isolate and identify SARS-coronavirus in nasal and throat swabs collected from clinically diagnosed severe acute respiratory syndrome (SARS) patients. METHODS: Nasal and throat swab specimens were inoculated onto well of 24-well plate containing confluent monolayers of Vero and MRC-5 cells. Isolates were identified with serology, electron microscopy and genome sequence. RESULTS: One hundred and fifty-eight nasal and throat swabs specimens from 79 SARS patients in Peking Union Medical College Hospital between April and May, 2003 were cultured for SARS-coronavirus. Cytopathic effect (CPE) was found in three nasal swab specimens inoculated in Vero cells. Acute and convalescent phase serum specimens collected from SARS patients were found with seroconversions and/or a fourfold or greater rises in indirect fluorescence antibodies (IgG and IgM) titers when the 3 isolates (infected Vero cells) were used as antigen. Coronavirus was observed in the culture supernatant by negative-stain electron microscopy. Genome sequence confirmed the isolates were SARS-coronavirus. CONCLUSIONS: The 3 isolates from nasal and throat swabs samples collected from 79 clinically diagnosed SARS patients were SARS coronavirus.

Identification of a novel PSR as the substrate of an SR protein kinase in the true slime mold.

Here, a novel cDNA encoding a serine/arginine (SR)-rich protein, designated PSR, was isolated from the true slime mold Physarum polycephalum and expressed in Escherichia coli. The deduced amino acid (aa) sequence reveals that PSR contains RS repeats at its C-terminus, similar to the conventional PSRPK substrate ASF/SF2. To study the novel protein, we generated a variety of mutant constructs by PCR and site-directed mutagenesis. Our analysis indicated that the purified recombinant PSR was phosphorylated by PSRPK in vitro and the SR-rich domain (amino acids 460-469) in the PSR protein was required for phosphorylation. In addition, removal of the docking motif (amino acids 424-450) from PSR significantly reduced the overall catalytic efficiency of the phosphorylation reaction. We also found that the conserved ATP-binding region (62)LGWGHFSTVWLAIDEKNGGREVALK(86) and the serine/threonine protein kinases active-site signature (184)IIHTDLKPENVLL(196) of PSRPK played a crucial role in substrate phosphorylation and Lys(86) and Asp(188) were crucial for PSRPK phosphorylation of PSR. These results suggest that PSR is a novel SR-related protein that is phosphorylated by PSRPK.

[Transient expression in microplasmodia of Physarum polycephalum].

The plasmodium of Physarum polycephalum is a suitable eukaryotic cell for cell cycle investigation, but there is no compatible transient expression system for the plasmodium. Using the promoter and terminator of ardC actin of Physarum polycephalum substituted the CMV IE and SV40 polyA of plasmid pDsRedl-N1, using cassette PardC-MCS-DsRed1-TardC substituted the cassette PardC-hph-TardC of plasmid pTB38, we constructed plasmids pXM1 and pXM2 for transient expression of red fluorescent protein (RFP) in Physarum polycephalum respectively. After reconstituting the transcription elongation factor homologous gene (pelf1) of Physarum polycephalum into the pXM2, we generated a plasmid pXM2-pelf1. After the plasmid pXM1, pXM2 and pXM2-pelf1 were electroporated into the plasmodium of Physarum polycephalum, we observed optimum RFP and PELF1-RFP expression under fluoroscope and confocal microscope between 24-48 h after electroporation, and found that ELF1-RFP expression was accumulated in nucleus of microplasmodium, the optimum electroporation parameters were 40 V/cm electric field, 1 ampere current, and 70 micros electric shock time. The results suggest that this expression system is qualified for transient expression of specific protein in plasmodium of Physarum polycephalum.

[Preparation and preliminary identification of the monoclonal antibody to bovine bFGF].

AIM: To prepare mAb against bovine bFGF and identify their Ig subgroups so as to establish an ELISA for detection of bFGF level. METHODS: BALB/c mice were immunized by recombinant bovine bFGF. Hybridoma cell lines which could stably secret the monoclonal antibodies to bFGF were established by cell fusion technique, and their related characteristics were identified. In addition, polyclonal antibodies to bFGF were prepared by immunization of rabbits with bovine bFGF. The mAb and polyclonal antibodies purified through protein A affinity chromatography were used to develop a sandwich ELISA for detection of bFGF level. RESULTS: Three hybridoma cell lines which could secret the mAbs IgG 1 to bFGF were obtained. The concentration of bFGF could be detected by sandwich ELISA developed with purified mAb and polyclonal antibody at nanogram level. CONCLUSION: mAb and polyclonal antibodies against to bovine bFGF have been prepared successfully, which provide powerful tool for further clinical application and related studies.