This study investigated the potential pathogenicity and genetic characteristics of ST314 Salmonella Kentucky(S.Ken-tucky)isolates from a broiler slaughterhouse.Antimicrobial susceptibility testing and whole-genome sequencing(WGS)were used to determine antimicrobial resistance,virulence factors,and the presence of antimicrobial resistance genes(ARGs)and mobile genetic elements(MGEs)among the isolates.The three multidrug resistant(MDR)isolates exhibited high resistance to multiple antimicrobial agents.The F4-2S strain exhibited resistance to 14 drugs across seven categories,whereas the F4T strain showed resistance to 13 drugs in the same number of categories.In contrast,the Y23 strain was resistant to nine drugs in six categories.Notably,F4-2S dem-onstrated high homology with F4T:both possessed 13 ARGs distributed across nine categories,in addition to a wide range of virulence factors,including secretion systems and effector proteins.The presence of IncR and IncX1 plasmids significantly enhanced both the antimicrobial resistance and pathogenicity of the isolates.The genome map of Y23 revealed a chromosome alongside two plasmids.The chromosome containedonly one resistance gene but several virulence factors,including the type III secretion system(T3SS),which is crucial for bacterial invasion.The plasmid pY23-1 contained eight types of 19 ARGs.Comparative analysis indicated that pY23-1 ex-hibited high homology with pZ1323SSL0055 and pSAL-045,all of which contained multiple ARGs,thus suggesting critical roles of these genes in the evolution of bacterial resistance.In conclusion,ST314 S.Kentucky demonstrated a complex mechanism of resis-tance coupled with significant pathogenic potential.The ARGs and MGEs in the plasmid contributed to the emergence and dissemina-tion of antimicrobial resistance.The multiple virulence factors present in the chromosome may be key factors driving the increasing virulence of ST314 S.Kentucky.

This study investigated the potential pathogenicity and genetic characteristics of ST314 Salmonella Kentucky(S.Ken-tucky)isolates from a broiler slaughterhouse.Antimicrobial susceptibility testing and whole-genome sequencing(WGS)were used to determine antimicrobial resistance,virulence factors,and the presence of antimicrobial resistance genes(ARGs)and mobile genetic elements(MGEs)among the isolates.The three multidrug resistant(MDR)isolates exhibited high resistance to multiple antimicrobial agents.The F4-2S strain exhibited resistance to 14 drugs across seven categories,whereas the F4T strain showed resistance to 13 drugs in the same number of categories.In contrast,the Y23 strain was resistant to nine drugs in six categories.Notably,F4-2S dem-onstrated high homology with F4T:both possessed 13 ARGs distributed across nine categories,in addition to a wide range of virulence factors,including secretion systems and effector proteins.The presence of IncR and IncX1 plasmids significantly enhanced both the antimicrobial resistance and pathogenicity of the isolates.The genome map of Y23 revealed a chromosome alongside two plasmids.The chromosome containedonly one resistance gene but several virulence factors,including the type III secretion system(T3SS),which is crucial for bacterial invasion.The plasmid pY23-1 contained eight types of 19 ARGs.Comparative analysis indicated that pY23-1 ex-hibited high homology with pZ1323SSL0055 and pSAL-045,all of which contained multiple ARGs,thus suggesting critical roles of these genes in the evolution of bacterial resistance.In conclusion,ST314 S.Kentucky demonstrated a complex mechanism of resis-tance coupled with significant pathogenic potential.The ARGs and MGEs in the plasmid contributed to the emergence and dissemina-tion of antimicrobial resistance.The multiple virulence factors present in the chromosome may be key factors driving the increasing virulence of ST314 S.Kentucky.

Aim To explore and verify the possible mechanism of Jiawei Duhuo Jisheng Mixture(JDJM)in the treatment on Knee Osteoarthritis(KOA)via using network pharmacology and animal experiment. Methods The ingredients of JDJM and relevant targets were collected from TCMSP and BATMAN-TCM database. The KOA-related targets were collected from GeneCard, OMIM and GEO databases. The common targets were acquired by intersecting ingredients-related and KOA-related targets, and then the Ingredient-Disease-Target Network and PPI network were constructed by Cytoscape 3.7.2 software and STRING platform. GO and KEGG enrichment analysis were performed based on Metascape database. Finally, the key targets and relevant mechanism were validated via animal experiment. Results In the network pharmacology study, 180 active ingredients related to treatment on KOA by JDJM were collected, and 152 common targets were confirmed. PPI network analysis showed that AKT1 might be the key targets of JDJM in the treatment on KOA. GO and KEGG enrichment analysis revealed that the key target mainly concentrated on inflammatory response and apoptosis. Animal experiment confirmed that JDJM could improve lesion in KOA rabbits, and suppress the expression levels of IL-1β, TNF-α, Caspase 3 and BAX in serum and articular fluid. AKT1 expression(including mRNA and protein)in articular cartilage was also down-regulated. Conclusions Based on the results of network pharmacology and animal experiment, JDJM may relieve KOA severity by anti-inflammatory and anti-apoptotic effects through a variety of molecular signaling pathways.

It is an urgent demand worldwide to control the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The 3-chymotrypsin-like protease (3CL^pro) and papain-like protease (PL^pro) are key targets to discover SARS-CoV-2 inhibitors. After screening 12 Chinese herbal medicines and 125 compounds from licorice, we found that a popular natural product schaftoside inhibited 3CL^pro and PL^pro with IC₅₀ values of 1.73 ± 0.22 and 3.91 ± 0.19 μmol/L, respectively, and inhibited SARS-CoV-2 virus in Vero E6 cells with EC₅₀ of 11.83 ± 3.23 μmol/L. Hydrogen-deuterium exchange mass spectrometry analysis, quantum mechanics/molecular mechanics calculations, together with site-directed mutagenesis indicated the antiviral activities of schaftoside were related with non-covalent interactions with H41, G143 and R188 of 3CL^pro, and K157, E167 and A246 of PL^pro. Moreover, proteomics analysis and cytokine assay revealed that schaftoside also regulated immune response and inflammation of the host cells. The anti-inflammatory activities of schaftoside were confirmed on lipopolysaccharide-induced acute lung injury mice. Schaftoside showed good safety and pharmacokinetic property, and could be a promising drug candidate for the prevention and treatment of COVID-19.

Objective: To summarize the clinical experience of expanded internal mammary artery perforator (IMAP) flap combined with vascular supercharge in reconstruction of faciocervical scar. Methods: The retrospective observational study was conducted. From September 2012 to May 2021, 23 patients with postburn or posttraumatic faciocervical scars who met the inclusion criteria were admitted to Shanghai Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine, including 18 males and 5 females, aged from 11 to 58 years, all of whom were reconstructed with expanded IMAP flaps. At the first stage, one or two skin and soft tissue expander (s) with appropriate rated capacity were implanted in the anterior chest area according to the location and size of the scars. The IMAP, thoracic branch of supraclavicular artery, and lateral thoracic artery were preserved during the operation. The skin and soft tissue expanders were inflated with normal saline after the operation. The flaps were transferred during the second stage. The dominant IMAP was determined preoperatively using color Doppler ultrasound (CDU) blood flow detector. The faciocervical scars were removed, forming wounds with areas of 9 cm×7 cm-28 cm×12 cm, and the perforators of superficial temporal artery and vein or facial artery and vein were preserved during the operation. The flaps were designed according to the area and size of the wounds after scar resection with the dominant IMAP as the pedicle. Single-pedicle IMAP flaps were used to repair small and medium-sized wounds. For larger defects, the blood perfusion areas of vessels in the anterior chest were evaluated by indocyanine green angiography (ICGA). In situations where the IMAP was insufficient to nourish the entire flap, double-pedicle flaps were designed by using the thoracic branch of supraclavicular artery or lateral thoracic artery for supercharging. Pedicled or free flap transfer was selected according to the distance between the donor areas and recipient areas. After transplantation of flaps, ICGA was conducted again to evaluate blood perfusion of the flaps. The donor sites of flaps were all closed by suturing directly. Statistics were recorded, including the number, rated capacity, normal saline injection volume, and expansion period of skin and soft tissue expanders, the location of the dominant IMAP, the total number of the flaps used, the number of flaps with different types of vascular pedicles, the flap area, the flap survival after the second stage surgery, the occurrence of common complications in the donor and recipient areas, and the condition of follow-up. Results: Totally 25 skin and soft tissue expanders were used in this group of patients, with rated capacity of 200-500 mL, normal saline injection volume of 855-2 055 mL, and expansion period of 4-16 months. The dominant IMAP was detected in the second intercostal space (20 sides) or the third intercostal space (5 sides) before surgery. A total of 25 expanded flaps were excised, including 2 pedicled IMAP flaps, 11 free IMAP flaps, 4 pedicled thoracic branch of supraclavicular artery+free IMAP flaps, and 8 free IMAP+lateral thoracic artery flaps, with flap areas of 10 cm×8 cm-30 cm×14 cm. After the second stage surgery, tip necrosis of flaps in three patients occurred, which healed after routine dressing changes; one patient developed arterial embolism and local torsion on the vascular pedicle at the anastomosis of IMAP and facial artery, and the blood supply recovered after thrombectomy and vascular re-anastomosis. Fourteen patients underwent flap thinning surgery in 1 month to 6 months after the second stage surgery. The follow-up for 4 months to 9 years showed that all patients had improved appearances of flaps and functions of face and neck and linear scar in the donor sites of flaps, and one female patient had obvious nipple displacement and bilateral breast asymmetry. Conclusions: The expanded IMAP flap is matched in color and texture with that of the face and neck, and its incision causes little damage to the chest donor sites. When combined with vascular supercharge, a double-pedicle flap can be designed flexibly to further enhance the blood supply and expand the flap incision area, which is a good choice for reconstruction of large faciocervical scar.
China ; Cicatrix/surgery* ; Female ; Humans ; Male ; Mammary Arteries/surgery* ; Perforator Flap ; Reconstructive Surgical Procedures ; Saline Solution ; Skin Transplantation ; Soft Tissue Injuries/surgery* ; Surgical Wound ; Treatment Outcome

Objective: To analyze the effects of transient receptor potential vanilloid type 4 (TRPV4) activation on the function and endothelial-to-mesenchymal transition (EndMT) of human umbilical vein endothelial cells (HUVECs), as well as to explore the effects of TRPV4 activation on blood perfusion and survival of rat perforator flap and the mechanism. Methods: The experimental research methods were used. The 3^rd to 6^th passages of HUVECs were used for experiments and divided into 0.5 μmol/L 4α-phorbol 12, 13-didecanoate (4αPDD) group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, 10.0 μmol/L 4αPDD group, and phosphate buffer solution (PBS) group, which were cultivated in corresponding final molarity of 4αPDD and PBS, respectively. The cell proliferation activity at 6 and 12 h of culture was detected using cell counting kit-8 (CCK-8). Another batch of cells was acquired and divided into PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group, which were treated similarly as described before and then detected for cell proliferation activity at 6, 12, 24, and 48 h of culture. The residual scratch area of cells at post scratch hour (PSH) 12, 24, and 48 was detected by scratch test, and the percentage of the residual scratch area was calculated. The number of migrated cells at 24 and 48 h of culture was detected by Transwell experiment. The tube-formation assay was used to measure the number of tubular structures at 4 and 8 h of culture. The protein expressions of E-cadherin, N-cadherin, Slug, and Snail at 24 h of culture were detected by Western blotting. All the sample numbers in each group at each time point in vitro experiments were 3. A total of 36 male Sprague-Dawley rats aged 8 to 10 weeks were divided into delayed flap group, 4αPDD group, and normal saline group according to the random number table, with 12 rats in each group, and iliolumbar artery perforator flap models on the back were constructed. The flap surgical delay procedure was only performed in the rats in delayed flap group one week before the flap transfer surgery. Neither rats in 4αPDD group nor normal saline group had flap surgical delay; instead, they were intraperitoneally injected with 4αPDD and an equivalent mass of normal saline, respectively, at 10 min before, 24 h after, and 48 h after the surgery. The general state of flap was observed on post surgery day (PSD) 0 (immediately), 1, 4, and 7. The flap survival rates were assessed on PSD 7. The flap blood perfusion was detected by laser speckle contrast imaging technique on PSD 1, 4, and 7. The microvascular density in the flap's choke vessel zone was detected by immunohistochemical staining. All the sample numbers in each group at each time point in vivo experiments were 12. Data were statistically analyzed with analysis of variance for factorial design, analysis of variance for repeated measurement, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: At 6 and 12 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 0.5 μmol/L 4αPDD group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, and 10.0 μmol/L 4αPDD group (P>0.05). At 6, 12, 24, and 48 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group (P>0.05). At PSH 12, the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (P>0.05). At PSH 24 and 48, compared with those in PBS group, the percentages of the residual scratch area of cells in 3 μmol/L 4αPDD group were significantly decreased (with t values of 2.83 and 2.79, respectively, P<0.05), while the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group showed no significant differences (P>0.05). At 24 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (P>0.05). At 48 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD groups were significantly greater than that in PBS group (with t values of 6.20 and 9.59, respectively, P<0.01). At 4 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly greater than that in PBS group (with t values of 4.68 and 4.95, respectively, P<0.05 or <0.01). At 8 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD and 3 μmol/L 4αPDD groups were similar to that in PBS group (P>0.05). At 24 h of culture, compared with those in PBS group, the protein expression level of E-cadherin of cells in 3 μmol/L 4αPDD group was significantly decreased (t=5.13, P<0.01), whereas there was no statistically significant difference in the protein expression level of E-cadherin of cells in 1 μmol/L 4αPDD group (P>0.05); the protein expression level of N-cadherin of cells in 3 μmol/L 4αPDD group was significantly increased (t=4.93, P<0.01), whereas there was no statistically significant difference in the protein expression level of N-cadherin of cells in 1 μmol/L 4αPDD group (P>0.05); the protein expression levels of Slug of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly increased (with t values of 3.85 and 6.52, respectively, P<0.05 or P<0.01); and the protein expression level of Snail of cells in 3 μmol/L 4αPDD group was significantly increased (t=4.08, P<0.05), whereas there was no statistically significant difference in the protein expression level of Snail of cells in 1 μmol/L 4αPDD group (P>0.05). There were no statistically significant differences in the protein expression levels of E-cadherin, N-cadherin, Slug, or Snail of cells between 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group (P>0.05). The general condition of flaps of rats in the three groups was good on PSD 0. On PSD 1, the flaps of rats in the three groups were basically similar, with bruising and swelling at the distal end. On PSD 4, the swelling of flaps of rats in the three groups subsided, and the distal end turned dark brown and necrosis occurred, with the area of necrosis in flaps of rats in normal saline group being larger than the areas in 4αPDD group and delayed flap group. On PSD 7, the necrotic areas of flaps of rats in the 3 groups were fairly stable, with the area of necrosis at the distal end of flap of rats in delayed flap group being the smallest. On PSD 7, the flap survival rates of rats in 4αPDD group ((80±13)%) and delayed flap group ((87±9)%) were similar (P>0.05), and both were significantly higher than (70±11)% in normal saline group (with t values of 2.24 and 3.65, respectively, P<0.05 or P<0.01). On PSD 1, the overall blood perfusion signals of rats in the 3 groups were basically the same, and the blood perfusion signals in the choke vessel zone were relatively strong, with a certain degree of underperfusion at the distal end. On PSD 4, the boundary between the surviving and necrotic areas of flaps of rats in the 3 groups became evident, and the blood perfusion signals in the choke vessel zone were improved, with the normal saline group's distal hypoperfused area of flap being larger than the areas in delayed flap group and 4αPDD group. On PSD 7, the blood perfusion signals of overall flap of rats had generally stabilized in the 3 groups, with the intensity of blood perfusion signal in the choke vessel zone and overall flap of rats in delayed flap group and 4αPDD group being significantly greater than that in normal saline group. On PSD 7, the microvascular density in the choke vessel zone of flap of rats in 4αPDD group and delayed flap group were similar (P>0.05), and both were significantly higher than that in normal saline group (with t values of 4.11 and 5.38, respectively, P<0.01). Conclusions: After activation, TRPV4 may promote the migration and tubular formation of human vascular endothelial cells via the EndMT pathway, leading to the enhanced blood perfusion of perforator flap and microvascular density in the choke vessel zone, and therefore increase the flap survival rate.
Animals ; Cadherins ; Endothelial Cells ; Humans ; Male ; Necrosis ; Perforator Flap ; Rats ; Rats, Sprague-Dawley ; Saline Solution ; TRPV Cation Channels

Aim To investigate the potential mechanism of Jiawei Duhuo Jisheng Mixture regulating intestinal flora in the treatment of knee osteoarthritis(KOA)by 16S rRNA sequencing.Methods Eight-week-old male C57 mice were randomly divided into three groups:sham group,DMM group,and model+Jiawei Duhuo Jisheng Mixture group(Mixture group),6 mice per group.KOA model was induced by destabilization of medial meniscus surgery.16.25 mL·kg-1 dose mixture was given daily to the mixture group,and normal saline was given to the sham and DMM group.After eight weeks,the knee joints and colons of mice were collected,and the knee joints were prepared into paraffin sections,and the cartilage changes were observed with Safranin O-Fast Green and immunohistochemistry staining.16S rRNA sequencing of intestinal contents was performed to observe the changes of intestinal flora.Results Compared with model group,Jiawei Duhuo Jisheng Mixture could significantly reduce cartilage wear and OARSI score(P=0.033 5,P=0.029 5).16S rRNA sequencing showed that Jiawei Duhuo Jisheng Mixtrue could change the intestinal flora richness of KOA model mice,and improve the Alpha diversity(Chao1,Simpson)and Beta diversity(PCoA,NMDS).LefSe analysis showed that there were species with significant difference in abundance among the three groups(P=0.001),mainly including Lactobacillus,Firmicutes,Proteobacteria and other species.MetaCyc analysis indicated that Jiawei Duhuo Jisheng Mixture had effects on various metabolic pathways such as fatty acid,sugar and amino acid of intestinal flora(P<0.05).Conclusions Jiawei Duhuo Jisheng Mixtrue can effectively protect the articular cartilage and delay the progression of KOA.The mechanism may be through regulating the intestinal flora structure,protecting the intestinal barrier and reducing the inflammatory response.

Aim To investigate the role of calcium-independent phospholipase A2(iPLA2)in calcium regu-lation of intrarenal artery smooth muscle contraction.Methods The method of measuring the tension of isolated arterioles was used to explore the effect of bromoenol lactone(BEL), a specific inhibitor of iPLA2, on the tension of the intrarenal arteries in mice induced by different calcium channels, and the laser confocal calcium measurement technology was used to investigate the effect of BEL on the intracellular calcium influx mediated by arachidonic acid-mediated calcium channels.Results The intrarenal artery concentration dependent contractile response induced by the vasoconstrictors phenylephrine and 5-hydroxy tryptamine was inhibited by BEL(P<0.01).The contraction curve induced by CaCl2 was also inhibited by BEL(P<0.05).In the calcium-free K-H solution incubated with nifedipine, the intrarenal artery vasoconstriction caused by the release of sarcoplasmic reticulum calcium and the calcium influx of the SOC channel induced by CaCl2 was inhibited by BEL(P<0.05).BEL significantly inhibited the external calcium influx mediated by the ARC channel of human aortic smooth muscle cell lines incubated with nifedipine(P<0.01).Conclusions iPLA2 mediates the contractile response of intrarenal arteries by regulating the functions of L-type calcium channels, sarcoplasmic reticulum calcium release, SOC channels and ARC channels.

Xiaoer-Feire-Kechuan (XFK) is an 11-herb Chinese medicine formula to treat cough and pulmonary inflammation.The complicated composition rendered its chemical analysis and effective-component elucidation.In this study,we combined quantitative analysis and bioactivity test to reveal the anti-inflammatory constituents of XFK.First,UPLC-DAD and UHPLC/Q-Orbitrap-MS methods were estab-lished and validated to quantify 35 analytes (covering 9 out of 11 herbs) in different XFK formulations.Parallel reaction monitoring mode built in Q-Orbitrap-MS was used to improve the sensitivity and selectivity.Then,anti-inflammatory activities of the 35 analytes were analyzed using in vitro COX-2 inhibition assay.Finally,major analytes forsythosides H,I,A (8-10),and baicalin (15) (total contents varied from 21.79 to 91.20 mg/dose in different formulations) with significant activities (inhibitory rate ≥ 80％) were proposed as the anti-inflammatory constituents of XFK.The present study provided an effective strategy to discover effective constituents of multi-herb formulas.

Objective To explore the endoscopic characteristics of multidrug-resistant tuberculosis (MDR-TB) combined with tracheobronchial tuberculosis (TBTB). Methods 248 MDR-TB as study group, they hospitalized from October 1st 2008 to June 31st, 2016. 274 cases of non MDR-TB with bacteria positive as control group over 2015, all of them received bronchoscopy, sputum cultured and drug sensitivity tested of Isoniazid and Rifampicin. We analyzed the results of bronchoscopy and demographic data. Results 248 cases of MDR-TB patients, of 175 (70.56%) were diagnosed TBTB by bronchoscopy, of 73 (29.44%) without TBTB. 274 cases of non MDR-TB with bacteria positive patients, of 146 (53.28%) were diagnosed TBTB, of 128 (46.72%) non TBTB, the difference of comparisons was statistically significant (χ2 = 16.42, P = 0.000). MDR-TB combined with TBTB median age was 32 years, non MDR-TB combined with TBTB median age 42 years, the difference was statistically significant (U = 9932.00, P = 0.001). Among the MDR-TB patients, of 75 (42.86%) TBTB in the upper right bronchial, of71 (40.57%) upper left bronchus, while non MDR-TB patients, of 70 (47.95%) and 60 (41.10%), there was no statistically significant difference (χ2 = 2.44, P = 0.786). Among the MDR-TB, of 76 (43.43%) were inflammation infiltration type, of 11 (6.29%) were necrosis type, of 13 cases (7.43%) granulation proliferative type, of 72 (41.14%) were scar stricture type, of 3 (1.71%) tube wall softening type. Among the non MDR-TB, in turn, TBTB type were 50 (34.25%), 41 (28.08%), 9 (6.16%), 40 (27.40%), 5 (3.43%), the difference were statistically significant (χ2 = 30.50, P = 0.000). Conclusions The detection rate of TBTB was higher in MDR-TB patients, that common occur in younger patients. TBTB common infringe on upper right bronchial and upper left bronchus, TBTB type most are inflammatory infiltration type and scar stricture type. More attention should be paid to bronchoscopy among MDR-TB patients.