Differential distribution of linezolid in diseased and nondiseased bones in patients with spinal tuberculosis.

BACKGROUND: Linezolid exhibits antibacterial activity against sensitive and drug-resistant strains of Mycobacterium tuberculosis. Knowledge on the distribution of linezolid in different types of bones in patients with spinal tuberculosis (TB) is lacking, which limits the pharmacokinetic and pharmacodynamic studies of linezolid. This study aimed to evaluate the distribution of linezolid in diseased and nondiseased bones in patients with spinal TB. METHODS: Spinal TB patients treated with linezolid-containing regimens and whose diseased and nondiseased bones were collected during surgery were enrolled retrospectively from January 2017 to February 2022. Blood, nondiseased bones, and diseased bones were collected simultaneously during the operation. Linezolid concentrations in the plasma, nondiseased bones, and diseased bones were subjected to high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Seven eligible spinal TB patients, including one rifampicin-resistant case, were enrolled. Following a 600 mg oral administration of linezolid before surgery, the median concentrations of linezolid in plasma, nondiseased bone, and diseased bone of the seven patients were 8.23, 1.01, and 2.13 mg/L, respectively. The mean ratios of linezolid concentration in nondiseased bones/plasma, diseased bones/plasma and diseased bones/nondiseased bones reached 0.26, 0.49, and 2.27, respectively. The diseased bones/plasma presented a higher mean ratio of linezolid concentration than nondiseased bones/plasma, and the difference was statistically significant (t = 2.55, p = 0.025). Pearson's correlation analysis showed the positively correlation of linezolid concentrations in diseased and nondiseased bones (r = 0.810, p = 0.027). CONCLUSIONS: Linezolid exhibits a higher concentration distribution in diseased bones than in nondiseased bones.

The Comparative Influence of 2 and 4 Weeks Preoperative Antituberculosis Treatment on Spinal Tuberculosis Surgery: A Multicenter, Prospective, Randomized Clinical Trial.

INTRODUCTION: A trade-off between successful surgery and minimizing the operation delay for patients with spinal tuberculosis (TB) is a major consideration to determine the duration of preoperational anti-TB treatment (AAT). In this study, 2 and 4 weeks preoperative AAT durations were compared for their influence on the operation outcomes. METHOD: A multicenter, prospective, randomized trial was conducted in four hospitals in China. New patients with spinal TB were recruited and randomly allocated to two groups (2 or 4 weeks' preoperative treatment) and administered the standardized first-line anti-TB drugs. The symptom changing and indicators reflecting recovery and side effects of the treatment were monitored. Patient was followed up for another 18 months after completion of treatment. RESULTS: In total, 150 eligible patients were enrolled between June 2014 and December 2016, and 13 patients were excluded after the enrollment. The remaining 137 participants were randomly allocated to the 2-week group (n = 68) or the 4-week group (n = 69). These two groups acquired similar surgical outcomes, considering wound healing rate within 3 months after the operation (94.20%, 65/69 vs 89.71%, 61/68; P = 0.333) and bony fusion rate within 6 months (98.46%, 64/65 vs 95.45%, 63/66; P = 0.317). However, the culture positive rate of pus collected during operation in the 4-week group (41.94%) was significantly lower than that of the 2-week group (60.94%, P = 0.033). No reoccurrence of disease was observed in either group during the 18-month follow-up period. CONCLUSION: Patients with spinal TB administered 2 or 4 weeks of preoperative anti-TB treatment acquired similar surgical outcomes. However, patients who underwent the operation sooner suffered 2 weeks less agony from the disease.

Novel compound heterozygous variants in the GFPT1 gene leading to rare limb-girdle congenital myasthenic syndrome with rimmed vacuoles.

BACKGROUND:  Congenital myasthenic syndrome (CMS) is a heterogeneous group of rare disorders with impaired neuromuscular transmission caused by genetic defects, which is characterized by fatigable muscle weakness. CASE PRESENTATION:  Herein, we report a case of limb-girdle CMS (LG-CMS) in a 15-year-old Chinese girl with limb weakness and mild ptosis. The patient presented with well-defined clinical manifestations, muscle imaging, and electrophysiological features associated with CMS. On muscle biopsy, in addition to tubular aggregates identified, an extremely unusual pathological change of rimmed vacuoles in muscle fibers was observed. Whole-exome sequencing disclosed two novel heterozygous variants (c.14 T>A and c.581 T>C) in the human glutamine-fructose-6-phosphate transaminase 1 (GFPT1) gene, leading to the substitutions of phenylalanine to tyrosine (p.F5Y) and serine (p.F194S), respectively. Both variants were predicted to be likely pathogenic by SIFT, Polyphen-2, and Mutation Taster. Treatments with pyridostigmine bromide and albuterol produced a dramatic improvement. CONCLUSIONS:  Collectively, molecular genetic analysis and muscle biopsy play crucial roles in the diagnosis of GFPT1-related LG-CMS with rimmed vacuoles (a rare phenotype of CMS) and have important implications for treatment decision.

CD3+, CD4+, and CD8+ Expression in Cells in Peripheral Blood of Silicosis Patients and Intervention Effect of Thymalfasin.

OBJECTIVE: This study aims to investigate the changes in CD3+, CD4+ and CD8+ expression in cells in peripheral blood of silicosis patients, and observe the immunoregulatory effect of thymalfasin. METHODS: A total of 80 silicosis patients were enrolled in the study, randomly divided into two groups: treatment group and control group (n=40, each group). In addition, 40 healthy adults, who underwent physical examinations in our hospital, were enrolled into the health examination group. Patients in the control group and treatment group were given anti-infection treatment, according to their conditions. Patients in the treatment group additionally received thymalfasin. Then, the number of peripheral blood T lymphocyte subsets in subjects in all three groups before and after treatment was measured. RESULTS: (1) Before treatment, CD3+, CD4+ and CD8+ levels in cells were significantly lower in the treatment group and control group than in the health examination group, and the differences were statistically significant (P<0.05). (2) In the treatment group, the number of CD4+ cells in peripheral blood was significantly higher after one week of treatment, when compared to that before treatment, and the difference was statistically significant (P<0.05). CONCLUSION: In silicosis patients, CD3+, CD4+ and CD8+ cells in peripheral blood are decreased, and thymalfasin can significantly increase CD4+ cells in peripheral blood of silicosis patients.

Diagnostic efficiency of Xpert MTB/RIF assay for osteoarticular tuberculosis in patients with inflammatory arthritis in China.

BACKGROUND: Both osteoarticular tuberculosis (OA-TB) and inflammatory arthritis can lead to osteoarticular structural damage. These conditions exhibit similar symptoms, physical signs, and imaging features. Rapidly and accurately diagnosing OA-TB in patients with inflammatory arthritis presents a challenge to clinicians. Xpert MTB/RIF (Xpert) has been endorsed by the World Health Organization (WHO) as a rapid diagnostic tool for diagnosis of pulmonary and extrapulmonary TB. This study was designed to investigate diagnostic efficiency of Xpert for OA-TB in patients with inflammatory arthritis in China. METHODS: A total of 83 consecutive patients with inflammatory arthritis and suspected OA-TB were enrolled prospectively from June 2014 to May 2018. Demographic, clinical, and biological data were recorded. Xpert assay, smear microscopy examination (smear), BACTEC MGIT 960 (MGIT 960), pathological examination, and T-SPOT.TB test were performed for each patient who received operations. Diagnostic efficiency of Xpert was evaluated based on a composite reference standard (CRS). RESULTS: A total of 49 out of 83 patients with inflammatory arthritis and suspected OA-TB received operations, and 49 specimens were obtained during operations. According to CRS, 36 out of 49 patients with inflammatory arthritis were diagnosed with OA-TB, and 13 were not affected by the condition. Sensitivity of Xpert assay, smear, MGIT 960, pathological examination, and T-SPOT.TB test reached 66.70% (24/36), 25.00% (9/36), 30.55% (11/36), 47.22% (17/36), and 80.55% (29/36), respectively. Specificity of Xpert assay, smear, MGIT 960, and pathological examination was all 100% (13/13). Specificity of T-SPOT.TB test was 53.84% (7/13). Sensitivity of Xpert was higher than that of smear, MGIT 960 and pathological examination, but the sensitivity of Xpert was lower than that of T-SPOT.TB. Sensitivity of Xpert was statistically different from that of smear and MGIT 960 (P<0.001, P = 0.002), but the sensitivity of Xpert was not significantly different from that of pathological examination and T-SPOT.TB (P = 0.096, P = 0.181). Specificity of T-SPOT.TB was less than that of Xpert, smear, MGIT 960, and pathological examination, and the difference between them was statistically significant (P = 0.015). Among the 27 OA-TB patients with smear negative results, Xpert had the highest sensitivity, but sensitivity of Xpert was not significantly different from that of pathological examination and T-SPOT.TB (P = 0.413, P = 0.783). 2 of 36 OA-TB patients exhibited RIF resistance. Xpert was concordant with MGIT 960-based drug susceptibility testing (DST) in detecting rifampin (RIF) resistance. CONCLUSIONS: Xpert is an efficient tool with high sensitivity and specificity for OA-TB diagnosis in patients with inflammatory arthritis in high-TB prevalence countries. Compared with conventional methods, Xpert has two advantages: one is fast, and the other is able to provide RIF resistance information simultaneously.

Transcriptional regulation of the Ufm1 conjugation system in response to disturbance of the endoplasmic reticulum homeostasis and inhibition of vesicle trafficking.

Homeostasis of the endoplasmic reticulum (ER) is essential for normal cellular functions. Disturbance of this homeostasis causes ER stress and activates the Unfolded Protein Response (UPR). The Ufm1 conjugation system is a novel Ubiquitin-like (Ubl) system whose physiological target(s) and biological functions remain largely undefined. Genetic study has demonstrated that the Ufm1-activating enzyme Uba5 is indispensible for erythroid differentiation in mice, highlighting the importance of this novel system in animal development. In this report we present the evidence for involvement of RCAD/Ufl1, a putative Ufm1-specific E3 ligase, and its binding partner C53/LZAP protein in ufmylation of endogenous Ufm1 targets. Moreover, we found that the Ufm1 system was transcriptionally up-regulated by disturbance of the ER homeostasis and inhibition of vesicle trafficking. Using luciferase reporter and ChIP assays, we dissected the Ufm1 promoter and found that Ufm1 was a potential target of Xbp-1, one of crucial transcription factors in UPR. We further examined the effect of Xbp-1 deficiency on the expression of the Ufm1 components. Interestingly, the expression of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type mouse embryonic fibroblasts (MEFs) was significantly induced by inhibition of vesicle trafficking, but the induction was negated by Xbp-1 deficiency. Finally, we found that knockdown of the Ufm1 system in U2OS cells triggered UPR and amplification of the ER network. Taken together, our study provided critical insight into the regulatory mechanism of the Ufm1 system and established a direct link between this novel Ubl system and the ER network.

A novel C53/LZAP-interacting protein regulates stability of C53/LZAP and DDRGK domain-containing Protein 1 (DDRGK1) and modulates NF-kappaB signaling.

C53/LZAP (also named as Cdk5rap3) is a putative tumor suppressor that plays important roles in multiple cell signaling pathways, including DNA damage response and NF-kappaB signaling. Yet how its function is regulated remains largely unclear. Here we report the isolation and characterization of two novel C53/LZAP-interacting proteins, RCAD (Regulator of C53/LZAP and DDRGK1) and DDRGK1 (DDRGK domain-containing protein 1). Our co-immunoprecipitation assays confirmed their interactions, while gel filtration assay indicated that C53/LZAP and RCAD may form a large protein complex. Intriguingly, we found that RCAD knockdown led to dramatic reduction of C53/LZAP and DDRGK1 proteins. We also found that C53/LZAP and DDRGK1 became more susceptible to the proteasome-mediated degradation in RCAD knockdown cells, whereas their ubiquitination was significantly attenuated by RCAD overexpression. In addition, we found that RCAD, like C53/LZAP, also plays an important role in regulation of NF-kappaB signaling and cell invasion. Taken together, our findings strongly suggest that RCAD is a novel regulator of C53/LZAP tumor suppressor and NF-kappaB signaling.

Effects on interactions of oppositely charged phospholipid vesicles of covalent attachment of polyethylene glycol oligomers to their surfaces: adhesion, hemifusion, full fusion and "endocytosis".

Oppositely charged giant vesicles are known to adhere, hemifuse and fuse, all of which depend upon the nature of surface contacts. To further understand such interactions, vesicles were surface-modified with polyethylene glycol (PEG), a moiety that reduces surface-surface interactions. Positively charged vesicles were composed of O-ethyldioleoylphosphocholine (EDOPC), dioleoylphosphatidylcholine (DOPC) and a carbocyanine dye (DiO), with and without DPPE-PEG (dipalmitoylphosphatidylethanolamine-N-PEG MW of the PEG portion = 2000). Negatively charged vesicles were composed of dioleoylphosphatidylglycerol (DOPG), DOPC and a rhodamine B dye (Rh-PE), with as well as without DPPE-PEG (MW 2,000). A microscope-mounted electrophoresis chamber allowed selected pairs of vesicles to be brought into contact while color images were collected at video rates (30 frames/s). Data collection focused on effects of PEG on vesicle interactions as a function of the surface charge density. Relative to PEG-free preparations, vesicles containing DPPE-PEG (1) formed larger contact zones, (2) underwent adhesion and fusion processes more slowly (by two to four times) and (3) at high charge density were less susceptible to rupture upon contact. Unexpectedly, PEG-containing vesicles exhibited engulfment of a smaller by a larger vesicle, a process topologically similar to cellular endocytosis. These observations are interpreted to mean that, although initial surface-surface interactions are weakened by the intervening layer of PEG chains, eventual and strong bilayer-bilayer contact is still possible, evidently because the lipid anchors of these chains can diffuse away from the contact zone.

Lipid bilayer vesicle fusion: intermediates captured by high-speed microfluorescence spectroscopy.

The fusion of lipid bilayers can be visualized under the fluorescence microscope, but the process is very fast and requires special techniques for its study. It is reported here that vesicle fusion is susceptible to analysis by microspectrofluorometry and that for the first time, the entire fusion process has been captured. In the case of giant (>10- micro m diameter) bilayer vesicles having a high density of opposite charge, fusion proceeds through stages of adhesion, flattening, hemifusion, elimination of the intervening septum, and uptake of excess membrane to generate a spherical product very rapidly. These investigations became possible with a fluorescence microscope that was modified for recording of images simultaneously with the collection of fluorescence emission spectra from many (>100) positions along the fusion axis. Positively-charged vesicles, composed of O-ethylphosphatidylcholine and dioleoylphosphatidylcholine, were labeled with a carbocyanine fluorophore. Negatively-charged vesicles, composed of dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine, were labeled with a rhodamine fluorophore that is a resonance energy transfer acceptor from the carbocyanine fluorophore. An electrophoretic chamber allowed selection of pairs of vesicles to be brought into contact and examined. Spectral changes along the axis of fusion were captured at high speed (a few ms/frame) by operating a sensitive digital camera in the virtual-chip mode, a software/hardware procedure that permits rapid readout of selected regions of interest and by pixel binning along the spectral direction. Simultaneously, color images were collected at video rates (30 frame/s). Comparison of the spectra and images revealed that vesicle fusion typically passes through a hemifusion stage and that the time from vesicle contact to fusion is <10 ms. Fluorescence spectra are well suited to rapid collection in the virtual-chip mode because spectra (in contrast to images) are accurately characterized with a relatively small number of points and interfering signals can be removed by judicious choice of barrier filters. The system should be especially well-suited to phenomena exhibiting rapid fluorescence change along an axis; under optimal conditions, it is possible to obtain sets of spectra (wavelength range of approximately 150 nm) at >100 positions along a line at rates >1000 frames/s with a spectral resolution of approximately 10 nm and spatial resolution at the limit of the light microscope ( approximately 0.2 micro m).