Characteristics and seasonal variations of cirrus clouds from ground-based lidar and satellite observations over Shouxian Area, China.

This study investigates the macroscopic and optical properties of cirrus clouds in the 32N region from July 2016 to May 2017, leveraging data from ground-based lidar observations and CALIOP to overcome the inconsistencies in detected cirrus cloud samples. Through extensive data analysis, statistical characteristics of cirrus clouds were discerned, revealing lidar ratio values of 28.5 ± 10.8 from ground-based lidar and 27.4 ± 11.2 from CALIOP. Validation with a decade of CALIOP data (2008-2018) confirmed these findings, presenting a consistent lidar ratio of 27.4 ± 12.0. A significant outcome of the analysis was the identification of a positive correlation between the lidar ratio and cloud centroid temperature, indicating a gradual decrease in the lidar ratio as temperatures dropped. The study established a fundamental consistency in their macroscopic properties, including cloud base height, cloud top height, cloud thickness, cloud centroid height, and cloud centroid temperature. The results for ground-based lidar (CALIOP) are: 10.0 ± 2.1 km (10.0 ± 2.2 km), 11.8 ± 2.1 km (11.5 ± 2.3 km), 1.87 ± 0.83 km (1.52 ± 0.71 km), and 10.5 ± 2.2 km, -46.9 ± 9.7°C (-47.1 ± 10.0°C).These properties exhibited seasonal variations, with cirrus clouds reaching higher altitudes in summer and lower in winter, influenced by the height of the tropopause. The optical properties of cirrus clouds were also analyzed, showing an annual average optical depth of 0.31 ± 0.35 for ground-based lidar and 0.32 ± 0.44 for CALIOP. The study highlighted the distribution of subvisible, thin, and thick cirrus clouds, with a notable prevalence of subvisible clouds during summer, suggesting their frequent formation above 14 km. Furthermore, the study observed linear growth in geometric thickness and optical depth up to 2.5 km from CALIOP and 2.9 km from ground-based lidar. Maximum optical depth was observed at cloud centroid temperatures of -35°C for CALIOP and -40°C for ground-based lidar, with optical depth decreasing as temperatures fell. This suggests that fully glaciated cirrus clouds exhibit the highest optical depth at warmer temperatures, within the complete glaciation temperature range of -35°C to -40°C.

A recombinant selective drug-resistant M. bovis BCG enhances the bactericidal activity of a second-line anti-tuberculosis regimen.

Drug-resistant tuberculosis (DR-TB) poses a new threat to global health; to improve the treatment outcome, therapeutic vaccines are considered the best chemotherapy adjuvants. Unfortunately, there is no therapeutic vaccine approved against DR-TB. Our study assessed the therapeutic efficacy of a recombinant drug-resistant BCG (RdrBCG) vaccine in DR-TB. We constructed the RdrBCG overexpressing Ag85B and Rv2628 by selecting drug-resistant BCG strains and transformed them with plasmid pEBCG or pIBCG to create RdrBCG-E and RdrBCG-I respectively. Following successful stability testing, we tested the vaccine's safety in severe combined immune deficient (SCID) mice that lack both T and B lymphocytes plus immunoglobulins. Finally, we evaluated the RdrBCG's therapeutic efficacy in BALB/c mice infected with rifampin-resistant M. tuberculosis and treated with a second-line anti-TB regimen. We obtained M. bovis strains which were resistant to several second-line drugs and M. tuberculosis resistant to rifampin. Notably, the exogenously inserted genes were lost in RdrBCG-E but remained stable in the RdrBCG-I both in vitro and in vivo. When administered adjunct to a second-line anti-TB regimen in a murine model of DR-TB, the RdrBCG-I lowered lung M. tuberculosis burden by 1 log10. Furthermore, vaccination with RdrBCG-I adjunct to chemotherapy minimized lung tissue pathology in mice. Most importantly, the RdrBCG-I showed almost the same virulence as its parent BCG Tice strain in SCID mice. Our findings suggested that the RdrBCG-I was stable, safe and effective as a therapeutic vaccine. Hence, the "recombinant" plus "drug-resistant" BCG strategy could be a useful concept for developing therapeutic vaccines against DR-TB.

Pyrazolo[1,5- a]pyridine Inhibitor of the Respiratory Cytochrome bcc Complex for the Treatment of Drug-Resistant Tuberculosis.

Respiration is a promising target for the development of new antimycobacterial agents, with a growing number of compounds in clinical development entering this target space. However, more candidate inhibitors are needed to expand the therapeutic options available for drug-resistant Mycobacterium tuberculosis infection. Here, we characterize a putative respiratory complex III (QcrB) inhibitor, TB47: a pyrazolo[1,5- a]pyridine-3-carboxamide. TB47 is active (MIC between 0.016 and 0.500 µg/mL) against a panel of 56 M. tuberculosis clinical isolates, including 37 multi-drug-resistant and two extensively drug-resistant strains. Pharmacokinetic and toxicity studies showed promising profiles, including negligible CYP450 interactions, cytotoxicity, and hERG channel inhibition. Consistent with other reported QcrB inhibitors, TB47 inhibits oxygen consumption only when the alternative oxidase, cytochrome bd, is deleted. A point mutation in the qcrB cd2-loop (H190Y, M. smegmatis numbering) rescues the inhibitory effects of TB47. Metabolomic profiling of TB47-treated M. tuberculosis H37Rv cultures revealed accumulation of steps in the TCA cycle and pentose phosphate pathway that are linked to reducing equivalents, suggesting that TB47 causes metabolic redox stress. In mouse infection models, a TB47 monotherapy was not bactericidal. However, TB47 was strongly synergistic with pyrazinamide and rifampicin, suggesting a promising role in combination therapies. We propose that TB47 is an effective lead compound for the development of novel tuberculosis chemotherapies.

Mutation EthAW21R confers co-resistance to prothionamide and ethionamide in both Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv.

Ethionamide (ETA) and prothionamide (PRO) are interchangeably used in tuberculosis (TB) chemotherapy regimens. Subtle discrepancies between biochemical and genetic information on the modes of sensitivity and resistance of isoniazid (INH) and ETA warrants further studies. We report a new mutation - EthAW21R - in Mycobacterium bovis Bacillus Calmette-Guérin that corresponds with co-resistance to both PRO and ETA, which to the best of our knowledge has not been reported before. Our findings suggest that mutation EthAW21R could be used as a marker site for testing PRO and ETA cross-resistance.

Pyrazinoic Acid Inhibits a Bifunctional Enzyme in Mycobacterium tuberculosis.

Pyrazinamide (PZA), an indispensable component of modern tuberculosis treatment, acts as a key sterilizing drug. While the mechanism of activation of this prodrug into pyrazinoic acid (POA) by Mycobacterium tuberculosis has been extensively studied, not all molecular determinants that confer resistance to this mysterious drug have been identified. Here, we report how a new PZA resistance determinant, the Asp67Asn substitution in Rv2783, confers M. tuberculosis resistance to PZA. Expression of the mutant allele but not the wild-type allele in M. tuberculosis recapitulates the PZA resistance observed in clinical isolates. In addition to catalyzing the metabolism of RNA and single-stranded DNA, Rv2783 also metabolized ppGpp, an important signal transducer involved in the stringent response in bacteria. All catalytic activities of the wild-type Rv2783 but not the mutant were significantly inhibited by POA. These results, which indicate that Rv2783 is a target of PZA, provide new insight into the molecular mechanism of the sterilizing activity of this drug and a basis for improving the molecular diagnosis of PZA resistance and developing evolved PZA derivatives to enhance its antituberculosis activity.

A Cassette Containing Thiostrepton, Gentamicin Resistance Genes, and dif sequences Is Effective in Construction of Recombinant Mycobacteria.

The genetic manipulation of Mycobacterium tuberculosis genome is limited by the availability of selection markers. Spontaneous resistance mutation rate of M. tuberculosis to the widely used kanamycin is relatively high which often leads to some false positive transformants. Due to the few available markers, we have created a cassette containing thiostrepton resistance gene (tsr) for selection in M. tuberculosis and M. bovis BCG, and gentamicin resistance gene (aacC1) for Escherichia coli and M. smegmatis mc2155, flanked with dif sequences recognized by the Xer system of mycobacteria. This cassette adds to the limited available selection markers for mycobacteria.

Progress in resistance mechanisms and diagnosis of non-first line anti-TB drugs.

Tuberculosis (TB) is one of the three major infectious diseases in China and all over the world. In 2014, for the first time, TB killed more people than HIV did. Non-first line anti-TB drugs are used as main drugs in the treatment of MDR-TB. However, MDR-TB can gradually develop as extensively drug-resistant TB (XDR-TB) because of poor diagnosis, the unreasonable treatment, poor medical conditions and so on. The death rate of XDR-TB is close to lung cancer. Research on the mechanism of drug resistance of Mycobacterium tuberculosis has turned to non first-line anti-TB drugs: second and third line drugs and some new anti-TB drugs in development. In this review, we summarized the drug resistance mechanisms of the common non-first line anti-TB drugs. Most of drug resistant TB patients can't get timely diagnosis and correct treatment. So at the end of this article, we also summarized the common methods to diagnose drug-resistant TB.

Identification of Lysine Acetylation in Mycobacterium abscessus Using LC-MS/MS after Immunoprecipitation.

Mycobacterium abscessus (MAB), which manifests in the pulmonary system, is one of the neglected causes of nontuberculous mycobacteria (NTM) infection. Treatment against MAB is difficult, characterized by its intrinsic antibiotic drug resistance. Lysine acetylation can alter the physiochemical property of proteins in living organisms. This study aimed to determine if this protein post-translational modification (PTM) exists in a clinical isolate M. abscessus GZ002. We used the antiacetyl-lysine immunoprecipitation to enrich the low-abundant PTM proteins, followed by the LC-MS/MS analysis. The lysine acetylome of M. abscessus GZ002 was determined. There were 459 lysine acetylation sites found in 289 acetylated proteins. Lysine acetylation occurred in 5.87% of the M. abscessus GZ002 proteome, and at least 25% of them were growth essential. Aerobic respiration and carbohydrate metabolic pathways of M. abscessus GZ002 were enriched with lysine acetylation. Through bioinformatics analysis, we identified four major acetyl motif logos (K(ac)Y, K(ac)F, K(ac)H, and DK(ac)). Further comparison of the reported M. tuberculosis (MTB) acetylomes and that of MAB GZ002 revealed several common features between these two species. The lysine residues of several antibiotic-resistance, virulence, and persistence-related proteins were acetylated in both MAB GZ002 and MTB. There were 51 identical acetylation sites in 37 proteins found in common between MAB GZ002 and MTB. Overall, we demonstrate a profile of lysine acetylation in MAB GZ002 proteome that shares similarities with MTB. Interventions that target at these conserved sections may be valuable as anti-NTM or anti-TB therapies.

Role of the Cys154Arg Substitution in Ribosomal Protein L3 in Oxazolidinone Resistance in Mycobacterium tuberculosis.

We expressed the wild-type rplC and mutated rplC (Cys154Arg) genes, respectively, in Mycobacterium tuberculosis H37Ra and H37Rv in an attempt to delineate the role of rplC (Cys154Arg) regarding oxazolidinone resistance. An increase of the MICs of linezolid (LZD) and sutezolid (PNU-100480, PNU) against the recombinant mycobacteria with overexpressed rplC mutation (Cys154Arg) was found, suggesting the rplC gene is a determinant of bacillary susceptibilities to LZD and PNU.

Design, Synthesis, and Biological Evaluation of Pyrazolo[1,5-a]pyridine-3-carboxamides as Novel Antitubercular Agents.

A series of pyrazolo[1,5-a]pyridine-3-carboxamide derivatives were designed and synthesized as new anti-Mycobacterium tuberculosis (Mtb) agents. The compounds exhibit promising in vitro potency with nanomolar MIC values against the drug susceptive H37Rv strain and a panel of clinically isolated multidrug-resistant Mtb (MDR-TB) strains. One of the representative compounds (5k) significantly reduces the bacterial burden in an autoluminescent H37Ra infected mouse model, suggesting its promising potential to be a lead compound for future antitubercular drug discovery.

Role of folP1 and folP2 Genes in the Action of Sulfamethoxazole and Trimethoprim Against Mycobacteria.

The combination of trimethoprim (TMP) and sulfamethoxazole (SMX) has been shown to be active against Mycobacterium tuberculosis (Mtb) in clinical tuberculosis (TB) treatment. However, the mechanism of action of TMP-SMX against Mtb is still unknown. To unravel this, we have studied the effect of TMP and SMX by deleting the folP2 gene in Mycobacterium smegmatis (Msm), and overexpressing the Mtb and Msm folP1/2 genes in Msm. Knocking out of the folP2 gene in Msm reduced the minimum inhibitory concentration of SMX 8-fold compared with wild type. Overexpression of the folP1 genes from Mtb and Msm increased the MICs by 4- and 2-fold in Msm for SMX and TMP, respectively. We show a strong correlation between the expression of folP1 and folP2 genes and TMP-SMX resistance in mycobacteria. This suggests that a combination of FolP2 inhibitor and SMX could be used for TB treatment with a better outcome.

Engineering more stable, selectable marker-free autoluminescent mycobacteria by one step.

In our previous study, we demonstrated that the use of the autoluminescent Mycobacterium tuberculosis as a reporter strain had the potential to drastically reduce the time, effort, animals and costs consumed in evaluation of the activities of drugs and vaccines in live mice. However, the strains were relatively unstable and lost reporter with time without selection. The kanamycin selection marker used wasn't the best choice as it provides resistance to amino glycosides which are an important class of second line drugs used in tuberculosis treatment. In addition, the marker could limit utility of the strains for screening of new potential drugs or evaluating drug combinations for tuberculosis treatment. Limited selection marker genes for mycobacterial genetic manipulation is a major drawback for such a marker-containing strain in many research fields. Therefore, selectable marker-free, more stable autoluminescent mycobacteria are highly needed. After trying several strategies, we created such mycobacterial strains successfully by using an integrative vector and removing both the resistance maker and integrase genes by Xer site-specific recombination in one step. The corresponding plasmid vectors developed in this study could be very convenient in constructing other selectable marker-free, more stable reporter mycobacteria with diverse applications.

Efficient construction of unmarked recombinant mycobacteria using an improved system.

The genetic study of mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium ulcerans, is hampered heavily by their slow growth. We have developed efficient, versatile, and improved genetic tools for constructing unmarked recombinant mycobacteria more rapidly including generating multiple mutants using the same antibiotic marker in both fast- and slow-growing mycobacteria.

FOS protein expression and role of the vagus nerve in the rat medullary visceral zone in multiple organ dysfunction syndrome caused by subarachnoid hemorrhage.

This study was designed to observe the role of FOS protein expression in the rat medullary visceral zone (MVZ) in multiple organ dysfunction syndrome (MODS) caused by subarachnoid hemorrhage (SAH), with and without severing the vagus nerve. We also investigated the regulatory and control mechanisms of the MVZ and the vagus nerve in MODS following SAH. A model of MODS following SAH was established by injecting arterial blood into the Willis' circle. The vagus nerve was cut off and blocked. The FOS protein expression in the MVZ was detected by immunohistochemistry. The positive expression levels of FOS in the MVZ in the SAH and SAH + severed-down vagus nerve (SDV) groups were higher than those in the normal control, sham surgery and SDV groups (P<0.01). However, expression in the SAH+SDV group was lower than that in the SAH group (P<0.01). Inflammatory damage was observed in each visceral organ at every time-phased point in the SAH group and the SAH+SDV group. The most apparent damage was at 24-36 h, consistent with the peak of FOS protein expression; the SAH+SDV group presented a greater level of damage. The inflammatory changes in surrounding visceral organs following SAH correlated with FOS protein expression in the MVZ, which indicates that the MVZ participates in the functional control of surrounding visceral organs following SAH. Severing the subphrenic vagus nerve increases the incidence of MODS following SAH and enhances SAH-induced inflammatory damage to the surrounding visceral organs, which indicates that the vagus nerve plays a role in the protection of the surrounding visceral organs in MODS following SAH.