Efficient biosynthesis of prunin in methanol cosolvent system by an organic solvent-tolerant α-L-rhamnosidase from Spirochaeta thermophila.

Prunin of desirable bioactivity and bioavailability can be transformed from plant-derived naringin by the key enzyme α-L-rhamnosidase. However, the production was limited by unsatisfactory properties of α-L-rhamnosidase such as thermostability and organic solvent tolerance. In this study, biochemical characteristics, and hydrolysis capacity of a novel α-L-rhamnosidase from Spirochaeta thermophila (St-Rha) were investigated, which was the first characterized α-L-rhamnosidase for Spirochaeta genus. St-Rha showed a higher substrate specificity towards naringin and exhibited excellent thermostability and methanol tolerance. The Km of St-Rha in the methanol cosolvent system was decreased 7.2-fold comparing that in the aqueous phase system, while kcat/Km value of St-Rha was enhanced 9.3-fold. Meanwhile, a preliminary conformational study was implemented through comparative molecular dynamics simulation analysis to explore the mechanism underlying the methanol tolerance of St-Rha for the first time. Furthermore, the catalytic ability of St-Rha for prunin preparation in the 20% methanol cosolvent system was explored, and 200 g/L naringin was transformed into 125.5 g/L prunin for 24 h reaction with a corresponding space-time yield of 5.2 g/L/h. These results indicated that St-Rha was a novel α-L-rhamnosidase suitable for hydrolyzing naringin in the methanol cosolvent system and provided a better alternative for improving the efficient production yield of prunin.

Dispelling Dampness, Relieving Turbidity and Dredging Collaterals Decoction, Attenuates Potassium Oxonate-Induced Hyperuricemia in Rat Models.

Purpose: Dispelling dampness, relieving turbidity and dredging collaterals decoction (DED), is a traditional Chinese medicine used in the treatment of hyperuricemia. We aimed to explore the effect and mechanism of DED in the treatment of hyperuricemia. Methods: The effects of DED (9.48, 4.74, and 2.37 g/kg/d) on potassium oxonate (750 mg/kg/d)-induced hyperuricemia in rats were evaluated by serum uric acid (UA), creatinine (CRE), blood urea nitrogen (BUN), and renal pathological changes. Network pharmacology was used to identify the effective components and targets of DED, and the key targets and signaling pathways for its effects on hyperuricemia were screened. Molecular docking was used to predict the action of DED. H&E, immunohistochemistry, WB, and PCR were used to validate the network pharmacology results. Results: DED can effectively alleviate hyperuricemia, inhibit UA, CRE, BUN, and xanthine oxidase (XOD) activity, and reduce renal inflammatory cell infiltration and glomerular atrophy. The experiment identified 27 potential targets of DED for hyperuricemia, involving 9 components: wogonin, stigmasterol 3-O-beta-D-glucopyranoside, 3ß-acetoxyatractylone, beta-sitosterol, stigmasterol, diosgenin, naringenin, astilbin, and quercetin. DED can relieve hyperuricemia mainly by inhibiting RAGE, HMGB1, IL17R, and phospho-TAK1, and by regulating the AGE-RAGE and IL-17 signaling pathways. Conclusion: DED can alleviate hyperuricemia by inhibiting XOD activity and suppressing renal cell apoptosis and inflammation via the AGE-RAGE signaling pathway and IL-17 signaling pathway. This study provides a theoretical basis for the clinical application of DED.

Design of Occupational Therapy Interventions for Middle-Aged and Elderly Family Caregivers.

This study aimed to develop an interactive app for occupational therapy interventions for middle-aged and elderly family caregivers by integrating life review and narrative therapy. The results indicated that the interventions improved the mood of individuals, but the improvement in the quality of life was less significant due to the multiple facets of life. The interface design of the interactive app had good operating characteristics and was above average in terms of learnability and usability. Overall, the intervention program positively improved participants' psychological aspect, which was in line with the purpose of the life review. Thus, a focus group put forward specific suggestions on the contextual construction of life review, the intelligent development of guiding questions, scenario simulation, and the introduction of creative collaboration functions, which could be used as a reference for subsequent content adjustment and interface design.

Misidentification of hepatic tuberculosis as cholangiocarcinoma: A case report.

BACKGROUND: Hepatic tuberculosis (TB) is uncommon clinically. Because of a lack of specific signs, characteristic symptoms and clinical manifestations and because pathological samples are difficult to obtain, hepatic TB is easily missed or misdiagnosed. CASE SUMMARY: A 62-year-old Chinese man presented with jaundice for 1 wk and no abnormal laboratory tests other than elevated bilirubin, aminotransferases and C-reactive protein. Computed tomography (CT) of the abdomen showed a mass in the left lobe of the liver and hepatic hilum with striped calcified foci. Mild enhancement was visible at the edges, along with extensive intrahepatic biliary ductal dilatation in the right lobe of the liver. In the arterial phase of both CT and magnetic resonance imaging, the main trunk and right branch of the portal artery were partially visualized. Magnetic resonance cholangiopancreatography (MRCP) indicated that the left lobe of the liver and most of the bile ducts in the hilum were not visible. Pathological examination revealed coagulative necrosis, and granulomatous nodules were seen around areas of necrosis; therefore, TB was considered. CONCLUSION: Hepatic tuberculosis is easily misdiagnosed or missed on imaging. Percutaneous puncture biopsy is the most useful tool for definitive diagnosis.

Development and evaluation of a novel personal sampler for PM1 using rotating porous media.

A rotating filtration device (RFD) that is filled with porous media is developed for sampling particulate matter with a diameter of 1 µm or less (PM1). An experiment is conducted on a test system using corn oil as a test aerosol. The RFD was evaluated using an aerodynamic particle sizer. The results yielded cutoff sizes of 1.68 µm and 1.04 µm at rotational speeds of 0 rpm and 9000 rpm, respectively, with foam with 80 pores per inch (ppi), a thickness of 80 mm, and a face velocity of 13.5 cm/s. When the foam was replaced with 94 ppi nickel mesh, the cutoff size decreased from 1.45 µm to 0.98 µm as the thickness increased from 17 to 68 mm. As the face velocity of the RFD increased from 13.5 to 108.3 cm/s, the cutoff size declined from 1.04 µm to 0.77 µm. Most of the changes in the cutoff sizes of the RFD were statistically significant (p < 0.05). The maximum pressure drops through the foam and the nickel mesh were 33.5 and 36.6 mm H2O, respectively. The experiment revealed that increasing the rotational speed and face velocity of the RFD, and the thickness and nominal size of the porous media reduced the penetration of oil aerosols.

Magnitude-dependent response of osteoblasts regulated by compressive stress.

The present study aimed to investigate the role of magnitude in adaptive response of osteoblasts exposed to compressive stress. Murine primary osteoblasts and MC3T3-E1 cells were exposed to compressive stress (0, 1, 2, 3, 4, and 5 g/cm2) in 3D culture. Cell viability was evaluated, and expression levels of Runx2, Alp, Ocn, Rankl, and Opg were examined. ALP activity in osteoblasts and TRAP activity in RAW264.7 cells co-cultured with MC3T3-E1 cells were assayed. Results showed that compressive stress within 5.0 g/cm2 did not influence cell viability. Both osteoblastic and osteoblast-regulated osteoclastic differentiation were enhanced at 2 g/cm2. An increase in stress above 2 g/cm2 did not enhance osteoblastic differentiation further but significantly inhibited osteoblast-regualted osteoclastic differentiation. This study suggested that compressive stress regulates osteoblastic and osteoclastic differentiation through osteoblasts in a magnitude-dependent manner.

Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways.

Lysophosphatidic acid (LPA) is an efficient, bioactive phospholipid involved in various biological processes. In this study, LPA-induced connective tissue growth factor (CTGF/CCN2) expression and the underlying mechanisms were investigated using the MC3T3-E1 cell line. The MC3T3-E1 cells were stimulated with an inhibitor of LPA receptors, an activator and inhibitor of protein kinase C (PKC) and protein kinase A (PKA) for indicated periods of time. RT-qPCR and western blot analyses were used to measure the expression levels of CCN2. Immunofluorescence staining was used to observe the translocation of PKC. The mRNA expression level of CCN2 was increased following stimulation of the cells with LPA; LPA transiently induced the mRNA expression of CCN2; maximum expression levels were observed 2 h following stimulation with LPA. This increase was accompanied by CCN2 protein synthesis. LPA receptor1/3 was inhibited by Ki16425, a specific inhibitor of LPA1/3; as a result, the LPA-induced increase in CCN2 expression was abrogated. LPA also induced the membrane translocation of PKC and enhanced PKC activity in the osteoblasts. Pre-treatment of the osteoblasts with staurosporine prevented the increase in CCN2 expression by induced by LPA, and the activation of PKC by phorbol 12-myristate 13-acetate (PMA) enhanced CCN2 expression, indicating that the PKC pathway is involved in the LPA-induced increase in CCN2 expression. The interference of PKA signaling also led to the induction of CCN2 expresion by LPA. These data indicate that LPA increases CCN2 expression through the activation of PKC and PKA. Thus, the regulatory functions of the PKC and PKA pathways are implicated in the LPA-induced increase in CCN2 expression.

Simultaneous detection of IgG and IgM antibodies against a recombinant polyprotein PstS1-LEP for tuberculosis diagnosis.

BACKGROUND: Commercial serological tests for the diagnosis of tuberculosis (TB) show poor sensitivity and specificity, and a new approach to antigen screening is required to improve the accuracy of serodiagnosis. METHODS: Using an indirect enzyme-linked immunosorbent assay (ELISA), we evaluated the responses of IgG and IgM antibodies to the recombinant PstS1-LEP protein expressed in Escherichia coli, a polyprotein of PstS1 and line multi-epitopes polypeptide (LEP). RESULTS: The mixture of anti-human IgG and IgM added to a well [Ig(G + M)], which was different from the combination of IgG and IgM (IgG + IgM), had a stronger immunoreactivity to PstS1-LEP than the single antibody. IgG and Ig(G + M), but not IgM against the PstS1-LEP protein effectively distinguished TB patients from patients with nontuberculous pulmonary disease (NTBPD) and healthy controls (HCs). Compared with IgG, the sensitivities of Ig(G + M) and IgG + IgM varied from 71.4% to 77.6% and 72.7% in pulmonary TB (PTB) patients and from 42.1% to 64.0% and 55.8% in extrapulmonary TB (EPTB) patients, respectively. The specificity of Ig(G + M) did not decrease, and was higher than that provided by IgG + IgM in HCs with positive tuberculin skin test. CONCLUSION: These findings suggest that PstS1-LEP can act as a candidate for detecting Ig(G + M) in serum from PTB and EPTB patients.

Assessment of five antigens from Mycobacterium tuberculosis for serodiagnosis of tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major public health issue, particularly in developing countries, and thus effective diagnostic methods for TB remain a central theme in basic and clinical research. To evaluate five antigens (38-kDa protein [38kDa], Rv3621c, Rv3618, 38kDa-ESAT-6 [38E6], and Ag85B-HBHA [AH]) in serological tests for TB patients, we recruited 288 patients and 201 healthy controls. The median IgG reactivity to 38kDa, 38E6, and AH was higher than that to Rv3618 and Rv3621c in pulmonary TB. 38kDa and 38E6 provided high sensitivities in pulmonary TB but low sensitivities in extrapulmonary TB (EPTB). The specificities achieved by 38kDa and 38E6 ranged from 82.0% to 93.9% in patients with non-TB respiratory disease (PD) and in controls. 38kDa and 38E6 exhibited lower sensitivities and higher specificities than their combinations with Rv3618. These findings provide useful information on the relative importance of the above five antigens and suggest that combinations of Rv3618 with 38kDa and 38E6 can increase their sensitivities, but their specificities need to be further increased.

Clinical evaluation of the recombinant 38 kDa protein of Mycobacterium tuberculosis.

The diagnosis of tuberculosis remains among public health concerns due to shortcomings of the purified protein derivative (PPD). Recombinant truncated 38 kDa protein (rTPA38) of Mycobacterium tuberculosis was evaluated to screen new tuberculosis-specific tuberculin. 539 patients, 1133 healthy controls, and 55 guinea pigs were recruited to assess their sensitivity and specificity to rTPA38; 221 healthy controls, with negative responses to rTPA38 and PPD, were vaccinated with M. bovis BCG to determine their cross-reactions with M. bovis BCG. The Mantoux technique was adopted to perform skin tests. No difference in the sensitivity of skin tests was detected between rTPA38 and PPD (78.2% vs 83.4%), but there was a significant difference in the specificity of skin tests between rTPA38 and PPD (75.2% vs 47.0%). Compared to PPD, rTPA38 elicited low positive responses for those recruitments vaccinated with M. bovis BCG. The rTPA38 had significant skin reactions in M. tuberculosis-sensitized guinea pigs, and the opposite was true for both M. fortuitum- and M. kansasii-sensitized guinea pigs. These findings indicate that rTPA38 may have potential as a tuberculosis-specific skin test antigen.

Extraction of antimicrobial peptides from pea protein hydrolysates by sulfonic acid functionalized biochar.

The extraction methods for antimicrobial peptides (AMPs) from plants are varied, but the absence of a standardized and rapid technique remains a challenge. In this study, a functionalized biochar was developed and characterized for the extraction of AMPs from pea protein hydrolysates. The results indicated that the biochar mainly enriched AMPs through electrostatic interaction, hydrogen bonding and pore filling. Then three novel cationic antimicrobial peptides were identified, among which the RDLFK (Arg-Asp-Leu-Phe-Lys) had the greatest inhibitory effect against Staphylococcus aureus and Bacillus subtilis, showcasing IC50 value of 2.372 and 1.000 mg/mL, respectively. Additionally, it was found that RDLFK could damage bacterial cell membranes and penetrate the cells to inhibit DNA synthesis. These results provided that the biochar-based extraction method presents an efficient and promising avenue for isolating AMPs, addressing a critical gap in the current methodologies for their extraction from plant sources.