GlmS plays a key role in the virulence factor expression and biofilm formation ability of Staphylococcus aureus promoted by advanced glycation end products.

Staphylococcus aureus (S. aureus) is well known for its biofilm formation ability and is responsible for serious, chronic refractory infections worldwide. We previously demonstrated that advanced glycation end products (AGEs), a hallmark of chronic hyperglycaemia in diabetic tissues, enhanced biofilm formation by promoting eDNA release via sigB upregulation in S. aureus, contributing to the high morbidity and mortality of patients presenting a diabetic foot ulcer infection. However, the exact regulatory network has not been completely described. Here, we used pull-down assay and LC-MS/MS to identify the GlmS as a candidate regulator of sigB in S. aureus stimulated by AGEs. Dual-luciferase assays and electrophoretic mobility shift assays (EMSAs) revealed that GlmS directly upregulated the transcriptional activity of sigB. We constructed NCTC 8325 ∆glmS for further validation. qRT-PCR analysis revealed that AGEs promoted both glmS and sigB expression in the NCTC 8325 strain but had no effect on NCTC 8325 ∆glmS. NCTC 8325 ∆glmS showed a significant attenuation in biofilm formation and virulence factor expression, accompanied by a decrease in sigB expression, even under AGE stimulation. All of the changes, including pigment deficiency, decreased haemolysis ability, downregulation of hla and hld expression, and less and sparser biofilms, indicated that sigB and biofilm formation ability no longer responded to AGEs in NCTC 8325 ∆glmS. Our data extend the understanding of GlmS in the global regulatory network of S. aureus and demonstrate a new mechanism by which AGEs can upregulate GlmS, which directly regulates sigB and plays a significant role in mediating biofilm formation and virulence factor expression.

[A case of trichoblastoma of the auricular cavity and review of the literature].

Trichoblastoma（TB） is a rare germ cell skin adnexal tumor of the hair, and it is a rare follicular tumor of the skin that differentiates from the hair germ epithelium and is often regarded as a benign skin tumorHowever, it is poorly confined and has a local infiltrative growth pattern. tb occurs in the head and neck region, especially in the face, and presents clinically as a slow growing, well-defined and elevated nodule. TB is routinely treated surgically. Due to the lack of universally accepted treatment guidelines or protocols, the recurrence rate after surgery is high, which makes clinical cure more difficult. In this study, a 65-year-old female patient was found to have a swelling with recurrent rupture and pus flow from the right external auditory canal opening and the auricular cavity. After initial misdiagnosis as otitis externa, she was treated with conventional anti-infective therapy, but her symptoms did not resolve and gradually worsened before coming to our hospital. The condition presented in this case is relativelyrare,therepre,timely and accurate diagnosis and treatment are crucial for prognosis improvement of such diseases.

Chemical profiling and rapid discrimination of Blumea riparia and Blumea megacephala by UPLC-Q-Exactive-MS/MS and HPLC.

Objective: To rapidly identify the two morphologies and chemical properties of similar herbal medicines, Blumea riparia and B. megacephala as the basis for chemical constituent analysis. Methods: UPLC-Q-Exactive-MS/MS was utilized for profiling and identification of the constituents in B. riparia and B. megacephala. Chemical pattern recognition (CPR) was further used to compare and distinguish the two herbs and to identify their potential characteristic markers. Then, an HPLC method was established for quality evaluation. Results: A total of 93 constituents are identified, including 54 phenolic acids, 35 flavonoids, two saccharides, one phenolic acid glycoside, and one other constituent, of which 67 were identified in B. riparia and B. megacephala for the first time. CPR indicates that B. riparia and B. megacephala samples can be distinguished from each other based on the LC-MS data. The isochlorogenic acid A to cryptochlorogenic acid peak area ratio calculated from the HPLC chromatograms was proposed as a differentiation index for distinguishing and quality control of B. riparia and B. megacephala. Conclusion: This study demonstrates significant differences between B. riparia and B. megacephala in terms of chemical composition. The results provide a rapid and simple strategy for the comparison and evaluation of the quality of B. riparia and B. megacephala.

De novo pyrimidine biosynthetic complexes support cancer cell proliferation and ferroptosis defence.

De novo pyrimidine biosynthesis is achieved by cytosolic carbamoyl-phosphate synthetase II, aspartate transcarbamylase and dihydroorotase (CAD) and uridine 5'-monophosphate synthase (UMPS), and mitochondrial dihydroorotate dehydrogenase (DHODH). However, how these enzymes are orchestrated remains enigmatical. Here we show that cytosolic glutamate oxaloacetate transaminase 1 clusters with CAD and UMPS, and this complex then connects with DHODH, which is mediated by the mitochondrial outer membrane protein voltage-dependent anion-selective channel protein 3. Therefore, these proteins form a multi-enzyme complex, named 'pyrimidinosome', involving AMP-activated protein kinase (AMPK) as a regulator. Activated AMPK dissociates from the complex to enhance pyrimidinosome assembly but inactivated UMPS, which promotes DHODH-mediated ferroptosis defence. Meanwhile, cancer cells with lower expression of AMPK are more reliant on pyrimidinosome-mediated UMP biosynthesis and more vulnerable to its inhibition. Our findings reveal the role of pyrimidinosome in regulating pyrimidine flux and ferroptosis, and suggest a pharmaceutical strategy of targeting pyrimidinosome in cancer treatment.

Glucose-6-phosphate dehydrogenase exerts antistress effects independently of its enzymatic activity.

G6PD (glucose-6-phosphate dehydrogenase) is the rate-limiting enzyme in the oxidative pentose phosphate pathway that can generate cytosolic NADPH for biosynthesis and oxidative defense. Since cytosolic NADPH can be compensatively produced by other sources, the enzymatic activity deficiency alleles of G6PD are well tolerated in somatic cells but the effect of null mutations is unclear. Herein, we show that G6PD KO sensitizes cells to the stresses induced by hydrogen peroxide, superoxide, hypoxia, and the inhibition of the electron transport chain. This effect can be completely reversed by the expressions of natural mutants associated with G6PD deficiency, even without dehydrogenase activity, exactly like the WT G6PD. Furthermore, we demonstrate that G6PD can physically interact with AMPK (AMPK-activated protein kinase) to facilitate its activity and directly bind to NAMPT (nicotinamide phosphoribosyltransferase) to promote its activity and maintain the NAD(P)H/NAD(P)+ homeostasis. These functions are necessary to the antistress ability of cells but independent of the dehydrogenase activity of G6PD. In addition, the WT G6PD and naturally inactive mutant also can similarly regulate the metabolism of glucose, glutamine, fatty acid synthesis, and GSH and interact with the involved enzymes. Therefore, our findings reveal the previously unidentified functions of G6PD that can act as the important physiological neutralizer of stresses independently of its enzymatic activity.

Disinfection Strategies for Carbapenem-Resistant Klebsiella pneumoniae in a Healthcare Facility.

Disinfectant resistance is evolving into a serious problem due to the long-term and extensive use of disinfectants, which brings great challenges to hospital infection control. As a notorious multidrug-resistant bacterium, carbapenem-resistant Klebsiella pneumoniae (CRKP) is one of the most common and difficult pathogens of nosocomial infection. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests of seven kinds of disinfectants (0.1% benzalkonium bromide, 4% aqueous chlorhexidine, 75% alcohol, entoiodine II, 2% glutaraldehyde, 2000 mg/L chlorine-containing disinfectants, and 3% hydrogen peroxide) were detected by the broth dilution method. Three efflux pump genes (oqxA, oqxB, and qacE∆1-sul1) were detected by PCR. The mean MIC value of aqueous chlorhexidine from the intensive care unit (ICU) (0.0034%) was significantly higher than that from non-ICUs (0.0019%) (p < 0.05). The positive rates of three efflux pump genes oqxA, oqxB and qacE∆1-sul1 were 60.9% (39/64), 17.2% (11/64) and 71.9% (46/64) in the detected CRKP isolates, respectively. This study discovered that CRKP strains demonstrated extensive resistance to clinical disinfectants and suggest that it is necessary to perform corresponding increases in the concentration of aqueous chlorhexidine and chlorine-containing disinfectants on the basis of current standards in the healthcare industry.

Reducing cadmium in rice using metallothionein surface-engineered bacteria WH16-1-MT.

Cadmium (Cd) accumulation in rice grains poses a health risk for humans. In this study, a bacterium, Alishewanella sp. WH16-1-MT, was engineered to express metallothionein on the cell surface. Compared with the parental WH16-1 strain, Cd2+ adsorption efficiency of WH16-1-MT in medium was increased from 1.2 to 2.6 mg/kg dry weight. The WH16-1-MT strain was then incubated with rice in moderately Cd-contaminated paddy soil. Compared with WH16-1, inoculation with WH16-1-MT increased plant height, panicle length and thousand-kernel weight, and decreased the levels of ascorbic acid and glutathione and the activity of peroxidase. Compared with WH16-1, WH16-1-MT inoculation significantly reduced the concentrations of Cd in brown rice, husks, roots and shoots by 44.0 %, 45.5 %, 36.1 % and 47.2 %, respectively. Moreover, inoculation with WH16-1-MT reduced the bioavailability of Cd in soil, with the total Cd proportion in oxidizable and residual states increased from 29 % to 32 %. Microbiome analysis demonstrated that the addition of WH16-1-MT did not significantly alter the original bacterial abundance and community structure in soil. These results indicate that WH16-1-MT can be used as a novel microbial treatment approach to reduce Cd in rice grown in moderately Cd-contaminated paddy soil.

Characterization of a novel extracellular CuZn superoxide dismutase from Rimicaris exoculata living around deep-sea hydrothermal vent.

Superoxide dismutase (SOD, EC 1.15.1.1) is a member of metalloenzyme that plays a key role in protecting organisms from oxidative damage. A novel extracellular CuZn superoxide dismutase RESOD was identified from Rimicaris exoculata, a dominant species that lives in close proximity to the deep-sea hydrothermal vents. It encoded a protein consisting of 227 amino acids with a signal peptide of 22 amino acids. Sequence analysis revealed that it had the characteristics of CuZn superoxide dismutase, and had low homology with the known SODs. Then the recombinant RESOD was expressed successfully, and high-purity RESOD was obtained. The recombinant RESOD exhibited maximal activity and stability with a temperature range of 0 °C to 10 °C. And the optimal pH for the activity and stability was about 10. However, RESOD was sensitive to some metal ions, particularly calcium. Furthermore, the biological function of RESOD was investigated in HeLa cells. It was found that RESOD could reduce the level of oxidation, and decrease the apoptosis resulted from excessive oxidant challenge. In conclusion, a novel alkali-tolerant cold-active extracellular CuZn SOD was characterized. The characteristics make RESOD a good candidate in a wide range of applications.

Carbon and nitrogen partitioning of transgenic rice T2A-1 (Cry2A*) with different nitrogen treatments.

Nitrogen (N) and carbon(C) metabolisms in plants were investigated to assess different responses of Bt and non-Bt rice to different N treatments. T2A-1 (Bt rice variety) inserted with Cry2A* protein to resist Lepidoptera and its parental line MH63 was adopted in this study. The total N accumulation presented no statistical difference. But nitrogen contents in different parts of rice plant were significantly different between the two lines, especially on leaf and spike part. This study revealed that the nitrogen in leaf of T2A-1 was far more than that of MH63; however, the nitrogen in spike of T2A-1 was less than that of MH63. In addition, MH63 assimilated more carbon than T2A-1. However, the distribution proportion of carbon in leaf, stem and spike of T2A-1 and MH63 were both 1:1:1. What's more, our study of the difference in metabolism pathway based on proteomics analysis provided more insights on the responses of two lines of Bt and non-Bt rice to different N treatments. And amino acid metabolism, energy metabolism, and carbohydrate metabolism presented significant difference between two lines. In addition, the number of differentially expressed proteins with N deficiency treatment was almost twice as many as that with normal N treatment. It could be inferred that the insertion of Cry2A* in T2A-1 may bring about effects on carbon and nitrogen allocation and related metabolisms, especially under N deficiency environment.

Phenylobacterium soli sp. nov., isolated from arsenic and cadmium contaminated farmland soil.

A Gram-stain-negative, facultative anaerobic, non-motile and rod-shaped bacterial strain, designated LX32T, was isolated from arsenic and cadmium contaminated farmland soil. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain LX32T was closely related to Phenylobacterium hankyongense HKS-05T (97.7 % sequence similarity), Phenylobacterium kunshanense CCTCC AB 2013085T (97.4 %) and Phenylobacterium deserti CCTCC AB 2016297T (97.1 %). The average nucleotide identity values of the whole genome sequences of LX32T/P. hankyongense HKS-05T, LX32T/P. kunshanense CCTCC AB 2013085T and LX32T/P. deserti CCTCC AB 2016297T were 79.8, 77.9 and 77.5 %, respectively. Its genome size was 4.02 Mb, comprising 3998 predicted genes with a DNA G+C content of 70.1 mol%. The major fatty acids were C15 : 0, C16 : 0 and summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c). The polar lipid profiles consisted of phosphatidylglycerol, aminophospholipid, seven glycolipids and two unidentified polar lipids. The predominantly respiratory quinone was ubiquinone-10. Based on polyphasic analyses, the isolate is considered to represent a novel species of the genus Phenylobacterium, for which the name Phenylobacterium soli sp. nov. is proposed. The type strain is LX32T (=KCTC 62522=CCTCC AB 2018055).

[Diagnostic value of dynamic-extended focused assessment with sonography for trauma in patients with multiple trauma].

OBJECTIVE: To investigate the diagnostic value of dynamic-extended focused assessment with sonography for trauma (D-EFAST) in patients with multiple trauma in intensive care unit (ICU). METHODS: A prospective clinical study was conducted. Eighty patients with multiple trauma admitted to ICU of Anhui Provincial Hospital from September 1st, 2014 to December 31st, 2016 were enrolled. Extended focused assessment with sonography for trauma (E-FAST) check was conducted at first, for those who had positive findings diagnosis was confirmed by immediately CT examination or surgical exploration. If it was negative, the patients received E-FAST every morning for 7 days (defined as D-EFAST), for those with positive findings, immediately CT or surgery was performed to clarify the diagnosis. The final clinical diagnosis was used as the "gold standard" to calculate the diagnostic accordance rate of EFAST and D-EFAST examination technique for pneumothorax, pleural effusion, spleen injury, kidney damage, liver damage, gastrointestinal injury, pericardial effusion, bladder rupture, and pancreatic injury, as well as their sensitivity, specificity, positive predictive value, negative predictive value, accuracy rate, and missed diagnosis rate, and the difference between EFAST and D-EFAST was compared. RESULTS: There were 4 patients excluded because of death and abandoning treatment, and finally 76 patients were included in the study. The total sensitivity of E-FAST examination technique for pneumothorax, pleural effusion, spleen injury, liver damage, gastrointestinal injury, pericardial effusion, and bladder rupture was 75.9% (66/87), and the specificity was 98.3% (587/597), the positive predictive value was 86.8% (66/76), and the negative predictive value was 96.5% (587/608), the accuracy rate was 95.5% (653/684), and the rate of missed diagnosis was 24.1% (21/87). The most of the delayed injury in patients with multiple trauma occurred at 2-7 days after injury with incidence of 4.8% (33/684). The diagnostic sensitivity of D-EFAST for delayed injury was 98.3% (118/120), the specificity was 99.8% (563/564), the positive predictive value was 99.2% (118/119), the negative predictive value was 99.6% (563/565), the diagnostic accuracy rate was 99.6% (681/684), and rate of missed diagnosis was 1.7% (2/120). When the final clinical diagnosis was set as the "gold standard", D-EFAST technology for the detection rate was 98.3% (118/120) for patients with multiple trauma on organ injury while the detection rate of E-FAST was 75.9% (66/87), with statistical significant difference (P < 0.01), indicating that D-EFAST was better than E-FAST in check of multiple trauma patients with organ injury. CONCLUSIONS: Although the E-FAST technology can quickly diagnose the multiple trauma patients and win the rescue time for critical patients, multiple trauma patients injured after 2-7 days prone to delayed damage and are difficult to detect, and D-EFAST can be used to find delayed damage earlier, and reduce the misdiagnosis rate of multiple trauma patients.

Cell proliferation and differentiation during the three dimensional reconstitution of eccrine sweat glands.

The aim of this study is to characterize the cell proliferation and proliferating cell types during three-dimensional reconstitution of eccrine sweat glands. Eccrine sweat gland cells suspended in Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 1, 2, 4, 6, 8, 14, 21, 28, 35 and 42 days post-implantation, Matrigel plugs were immunostained for Ki67, to detect cycling cells, and the Ki67 labeling index at different time points was calculated. Three pairs of antibodies, Ki67/K7, Ki67/K14 and Ki67/α-SMA, were used to identify proliferating cell types in the plugs, on days 28, 35 and 42, by immunofluorescence double staining. The Ki67 labeling index on the first day of implantation was 30.53%, rapidly reached a peak value of 81.43% at 2 days post-implantation, and then decreased gradually to a low of 2.87% at 42 days. Double immunofluorescence staining showed that K14/Ki67 double-stained cells accounted for 80% of the Ki67-positive cells, whereas K7/Ki67 and α-SMA/Ki67 double-stained cells each accounted for 10% of the Ki67-positive population on days 28, 35, or 42 post-implantation. We conclude that eccrine sweat gland cells rapidly enter the cell cycle after implantation, but quickly show decreased cell proliferation and increased cell differentiation.

Human eccrine sweat gland cells reconstitute polarized spheroids when subcutaneously implanted with Matrigel in nude mice.

Increasing evidence indicates that maintenance of cell polarity plays a pivotal role in the regulation of glandular homeostasis and function. We examine the markers for polarity at different time points to investigate the formation of cell polarity during 3D reconstitution of eccrine sweat glands. Mixtures of eccrine sweat gland cells and Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 2, 3, 4, 5 and 6 weeks post-implantation, Matrigel plugs were removed and immunostained for basal collagen IV, lateral ß-catenin, lateroapical ZO-1 and apical F-actin. The results showed that the cell polarity of the spheroids appeared in sequence. Formation of basal polarity was prior to lateral, apical and lateroapical polarity. Collagen IV was detected basally at 2 weeks, ß-catenin laterally and ZO-1 lateroapically at 3 weeks, and F-actin apically at 4 weeks post-implantation. At week 5 and week 6, the localization and the positive percentage of collagen IV, ß-catenin, ZO-1 or F-actin in spheroids was similar to that in native eccrine sweat glands. We conclude that the reconstituted 3D eccrine sweat glands are functional or potentially functional.

Changes in keratins and alpha-smooth muscle actin during three-dimensional reconstitution of eccrine sweat glands.

We have examined the changes of keratins and alpha-SMA at various time points in order to investigate the development and differentiation of eccrine sweat gland cells during the course of three-dimensional (3D) reconstitution. Mixtures of eccrine sweat gland cells and Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 1, 2, 4, 6, 8, 14, 21, 28, 35, and 42 days post-implantation, Matrigel plugs were removed and immunostained. We found that during 3D reconstitution, keratin and alpha-SMA expression changed in a time-dependent manner. At day 1, all cells stained positively for keratin isoforms K5, K14, and K15, with the staining intensity of K15 being weak and K5 and K14 being strong, but none of the cells displayed K7, K8, or alpha-SMA. As time progressed, spheroid-like structures formed with the inner layer acquiring K7 and K8, but losing K5 and K14 expression, and the outer layer acquiring alpha-SMA expression, but losing K15 expression. K8 expression was first noted at day 14, and K7 and alpha-SMA at day 21. The loss of K15 expression was first noted at day 14, K14 at day 21, and K5 at day 28. At 28, 35, and 42 days, the spheroid-like structures could be distinguished, by immunohistochemistry, as having secretory coil-like and coiled duct-like structures. We conclude that the changes in expression of keratins and alpha-SMA in 3D-reconstituted eccrine sweat glands are similar to those of native eccrine sweat glands, indicating that the 3D reconstitution of sweat glands provides an excellent model for studying the development, cytodifferentiation, and regulation of eccrine sweat glands.

BrdU-label-retaining cells in rat eccrine sweat glands over time.

Cell proliferation and turnover are fueled by stem cells. In a previous study, we demonstrated that rat eccrine sweat glands contained abundant bromodeoxyuridine (BrdU)-label-retaining cells (LRCs). However, morphological observations showed that eccrine sweat glands usually show little or no signs of homeostatic change. In this study, we account for why the homeostatic change is rare in eccrine sweat glands based on cytokinetic changes in BrdU-LRC turnover, and also determine the BrdU-labeled cell type. Thirty-six newborn SD rats, were injected intraperitoneally with 50mg/kg BrdU twice daily at a 2h interval for 4 consecutive days. After a chase period of 4, 6, 8, 12, 24 and 32 weeks, rats were euthanized, and the hind footpads were removed and processed for BrdU immunostaining, and BrdU/α-SMA and BrdU/K14 double-immunostaining. BrdU-LRCs were observed in the ducts, secretory coils and mesenchymal cells at all survival time points. The percentage of BrdU(+) cells in rat eccrine sweat glands averaged 4.2±1.2% after 4 weeks of chase, increased slightly by the 6th week, averaging 4.4±0.9%, and peaked at 8 weeks, averaging 5.3±1.0%. Subsequently, the average percentage of BrdU(+) cells declined to 3.2±0.8% by the 32nd week. There was no difference in the percentage of BrdU-LRCs among the different survival time points except that a significant difference in the percentage of BrdU-LRCs detected at 24 weeks versus 8 weeks, and 32 weeks versus 8 weeks, was observed. We concluded that the BrdU-LRCs turnover is slow in eccrine sweat glands.

Three-dimensional co-culture of BM-MSCs and eccrine sweat gland cells in Matrigel promotes transdifferentiation of BM-MSCs.

Victims with extensive and deep burns are unable to regenerate eccrine sweat glands. Combining of stem cells and biomimetic ECM to generate cell-based 3D tissues is showing promise for tissue repair and regeneration. We co-cultured BrdU-labeled bone marrow-derived mesenchymal stem cells (BM-MSCs) and eccrine sweat gland cells in Matrigel for 2 weeks in vitro and then evaluated for BM-MSCs differentiation into functional eccrine sweat gland cells by morphological assessment and immunohistochemical double staining for BrdU/pancytokeratin, BrdU/ZO-2, BrdU/E-cadherin, BrdU/desmoglein-2, BrdU/Na(+)-K(+)-ATPase α, BrdU/NHE1 and BrdU/CFTR. Cells formed spheroid-like structures in Matrigel, and BrdU-labeled BM-MSCs were involved in the 3D reconstitution of eccrine sweat gland tissues, and the incorporated BM-MSCs expressed an epithelial cell marker (pancytokeratin), epithelial cell junction proteins (ZO-2, E-cadherin and desmoglein-2) and functional proteins of eccrine sweat glands (Na(+)-K(+)-ATPase α, NHE1 and CFTR). In conclusion, three-dimensional co-culture of BM-MSCs and eccrine sweat gland cells in Matrigel promotes the transdifferentiation of BM-MSCs into potentially functional eccrine sweat gland cells.

Combination of keratins and alpha-smooth muscle actin distinguishes secretory coils from ducts of eccrine sweat glands.

Eccrine sweat glands are comprised of secretory coils and ducts, which are distinct in morphology and function. To better understand the roles of the two parts in development, homeostasis, wound repair and regeneration of eccrine sweat glands, we must distinguish between them. In this study, the localization of keratins and alpha-SMA in human eccrine sweat glands was examined by immunofluorescence staining. Based on the differential localization of keratins and alpha-SMA in different cell types, four pairs of antibodies (K5/K7, K5/alpha-SMA, K14/K7 and K14/alpha-SMA) were used to differentiate secretory coils from ducts by double-immunofluorescence staining. Immunofluorescence staining showed that myoepithelial cells of secretory coils expressed K5, K14 and alpha-SMA, whereas secretory cells of secretory coils expressed K7, K8, K15, K18 and K19. Ductal cells expressed K5, K8, K14 and K19. Double-staining showed that the secretory coils were K5(+)/K7(+), K5(+)/alpha-SMA(+), K14(+)/K7(+) and K14(+)/alpha-SMA(+), whereas ducts were K5(+)/K7(-), K5(+)/alpha-SMA(-), K14(+)/K7(-) and K14(+)/alpha-SMA(-). In conclusion, by combining use of keratins and alpha-SMA antibodies, secretory coils can be easily differentiated from ducts in morphology.

Matrigel basement membrane matrix induces eccrine sweat gland cells to reconstitute sweat gland-like structures in nude mice.

BACKGROUND: Severe burn results in irreversible damage to eccrine sweat glands, for which no effective treatment is available. Interaction between the extracellular matrix and epithelial cells is critical for proper three-dimensional organization and function of the epithelium. METHODS: Matrigel-embedded eccrine sweat gland cells were subcutaneously implanted into the inguinal regions of nude mice. Two weeks later, the Matrigel plugs were removed and evaluated for series of detection items. RESULTS: Sweat gland cells developed into sweat gland-like structures in the Matrigel plugs based on: (1) de novo formation of tubular-like structures with one or more hollow lumens, (2) expression of epithelial and sweat gland markers (pancytokeratin, CK5/7/14/19, α-SMA and CEA), (3) basement membrane formation, (4) myoepithelial cells presenting in and encompassing the tubular-like structures, (5) cellular polarization, evident by the expression of tight junction proteins (claudin-1 and ZO-2), anchoring junctions (desmoglein-1 and -2 and E-cadherin) and CEA in the luminal membrane, (6) expression of proteins related to sweat secretion and absorption (Na(+)-K(+)-ATPase α/ß, Na(+)-K(+)-2Cl-cotranspoter 1, Na(+)/H(+) exchanger 1, aquaporin-5, epithelial sodium channel, cystic fibrosis transmembrane conductance regulator, potassium channel and vacuolar-type H+-ATPase), and (7) about 20% of the tubular-like structures are de novo coils and 80% are de novo ducts. CONCLUSIONS: This study provides not only an excellent model to study eccrine sweat gland development, cytodifferentiation and reconstitution, but also an in vivo model for regeneration of eccrine sweat glands.

Localization of Na(+)-K(+)-ATPase α/ß, Na(+)-K(+)-2Cl-cotransporter 1 and aquaporin-5 in human eccrine sweat glands.

In order to evaluate the function of the repaired or regenerated eccrine sweat glands, we must first localize the proteins involved in sweat secretion and absorption in normal human eccrine sweat glands. In our studies, the cellular localization of Na(+)-K(+)-ATPase α/ß, Na(+)-K(+)-2Cl-cotransporter 1 (NKCC1) and aquaporin-5 (AQP5) in eccrine sweat glands were detected by immunoperoxidase labeling. The results showed that Na(+)-K(+)-ATPase α was immunolocalized in the cell membrane of the basal layer and suprabasal layer cells of the epidermis, the basolateral membrane of the secretory coils, and the cell membrane of the outer cells and the basolateral membrane of the luminal cells of the ducts. The localization of Na(+)-K(+)-ATPase ß in the secretory coils was the same as Na(+)-K(+)-ATPase α, but Na(+)-K(+)-ATPase ß labeling was absent in the straight ducts and epidermis. NKCC1 labeling was seen only in the basolateral membrane of the secretory coils. AQP5 was strongly localized in the apical membrane and weakly localized in the cytoplasm of secretory epithelial cells. The different distribution of these proteins in eccrine sweat glands was related to their functions in sweat secretion and absorption.

The cellular localization of Na(+)/H(+) exchanger 1, cystic fibrosis transmembrane conductance regulator, potassium channel, epithelial sodium channel γ and vacuolar-type H+-ATPase in human eccrine sweat glands.

The secretory portions of human eccrine sweat glands secrete isotonic fluid into the lumen and then the primary fluid is rendered hypotonic during its passage to the skin surface. During the processes of sweat secretion and absorption, many enzymes and proteins play important roles. In the study, the cellular localizations of Na(+)/H(+) exchanger 1 (NHE1), cystic fibrosis transmembrane conductance regulator (CFTR), potassium channel (KC), epithelial sodium channel γ (γENaC) and vacuolar-type H+-ATPase (V-ATPase) in human eccrine sweat glands and epidermis were detected using immunofluorescence labeling. The results revealed that in the secretory coils, the basolateral membranes showed evidence of CFTR, NHE1 and KC activities, the apical membranes showed the activities of KC and NHE1, and the nucleus showed γEaNC and V-ATPase activities; in the duct, the peripheral and luminal ductal cells showed evidence of CFTR, NHE1 and KC, the apical membranes showed the activities of CFTR and NHE1, and the nucleus showed γEaNC, V-ATPase and KC activities. The cellular localization of these proteins in eccrine sweat glands is helpful to better understand the mechanisms of sweat secretion and absorption.