From immunology to artificial intelligence: revolutionizing latent tuberculosis infection diagnosis with machine learning.

Latent tuberculosis infection (LTBI) has become a major source of active tuberculosis (ATB). Although the tuberculin skin test and interferon-gamma release assay can be used to diagnose LTBI, these methods can only differentiate infected individuals from healthy ones but cannot discriminate between LTBI and ATB. Thus, the diagnosis of LTBI faces many challenges, such as the lack of effective biomarkers from Mycobacterium tuberculosis (MTB) for distinguishing LTBI, the low diagnostic efficacy of biomarkers derived from the human host, and the absence of a gold standard to differentiate between LTBI and ATB. Sputum culture, as the gold standard for diagnosing tuberculosis, is time-consuming and cannot distinguish between ATB and LTBI. In this article, we review the pathogenesis of MTB and the immune mechanisms of the host in LTBI, including the innate and adaptive immune responses, multiple immune evasion mechanisms of MTB, and epigenetic regulation. Based on this knowledge, we summarize the current status and challenges in diagnosing LTBI and present the application of machine learning (ML) in LTBI diagnosis, as well as the advantages and limitations of ML in this context. Finally, we discuss the future development directions of ML applied to LTBI diagnosis.

Novel Mechanism for Cyclic Dinucleotide Degradation Revealed by Structural Studies of Vibrio Phosphodiesterase V-cGAP3.

3'3'-cyclic GMP-AMP (3'3'-cGAMP) belongs to a family of the bacterial secondary messenger cyclic dinucleotides. It was first discovered in the Vibrio cholerae seventh pandemic strains and is involved in efficient intestinal colonization and chemotaxis regulation. Phosphodiesterases (PDEs) that degrade 3'3'-cGAMP play important regulatory roles in the relevant signaling pathways, and a previous study has identified three PDEs in V. cholerae, namely, V-cGAP1, V-cGAP2, and V-cGAP3, functioning in 3'3'-cGAMP degradation. We report the crystal structure, biochemical, and structural analyses of V-cGAP3, providing a foundation for understanding the mechanism of 3'3'-cGAMP degradation and regulation in general. Our crystal and molecular dynamic (MD)-simulated structures revealed that V-cGAP3 contains tandem HD-GYP domains within its N- and C-terminal domains, with similar three-dimensional topologies despite their low-sequence identity. Biochemical and structural analyses showed that the N-terminal domain plays a mechanism of positive regulation for the catalytic C-terminal domain. We also demonstrated that the other homologous Vibrio PDEs, V-cGAP1/2, likely function via a similar mechanism.

Single Mutations Reshape the Structural Correlation Network of the DMXAA-Human STING Complex.

Subtle changes in protein sequences are able to alter ligand-protein interactions. Unraveling the mechanism of such phenomena is important for understanding ligand-protein interactions, including the DMXAA-STING interaction. DMXAA specifically binds to mouse STING instead of human STING. However, the S162A mutation and a newly discovered E260I mutation endow human STINGAQ with DMXAA sensitivity. Through molecular dynamics simulations, we revealed how these single mutations alter the DMXAA-STING interaction. Compared to mutated systems, structural correlations in the interaction of STINGAQ with DMXAA are stronger, and the correlations are cross-protomers in the dimeric protein. Analyses on correlation coefficients lead to the identification of two key interactions that mediate the strong cross-protomer correlation in the DMXAA-STINGAQ interaction network: DMXAA-267T-162S* and 238R-260E*. These two interactions are partially and totally interrupted by the S162A and E260I mutations, respectively. Moreover, a smaller number of water molecules are displaced upon DMXAA binding to STINGAQ than that on binding to its mutants, leading to a larger entropic penalty for the former. Considering the sensitivity of STINGAQ and two of its mutants to DMXAA, a strong structural correlation appears to discourage DMXAA-STING binding. Such an observation suggests that DMXAA derivatives, which are deprived of hydrogen-bond interaction with both 162S* and 267T, are potential agonists of human STING.

Rat and human STINGs profile similarly towards anticancer/antiviral compounds.

Cyclic dinucleotides (CDNs) and antitumor/antiviral agents (DMXAA and CMA) trigger STING-dependent innate immunity activation. Accumulative evidences have showed that DMXAA and CMA selectively activate mouse, but not human STING signaling. The mechanism underlying this species selectivity remains poorly understood. In this report, we have shown that human and rat STINGs display more similar signaling profiles toward DMXAA and CMA than that of human and mouse STINGs, suggesting that rat is more suitable for preclinical testing of STING-targeted drugs. We have also determined the crystal structures of both apo rat STING and its complex with cyclic GMP-AMP with 2'5' and 3'5' phosphodiester linkage (2'3'-cGAMP), a human endogenous CDN. Structure-guided biochemical analysis also revealed the functional importance of the connecting loop (A140-N152) between membrane and cytosolic domains in STING activation. Taken together, these findings reveal that rat STING is more closely related to human STING in terms of substrate preference, serving as a foundation for the development of STING-targeted drugs.

BMP-7 improved proliferation and hematopoietic reconstitution potential of ex vivo expanded cord blood-derived CD34(+) cells.

Due to limited availability, ex vivo expansion is essential for clinical applications of hematopoietic stem cells (HSCs). Bone morphogenetic proteins (BMPs) play an important role in regulating hematopoiesis development. In this study, the effects of BMP-2 and BMP-7 at different doses on expansion, clonogenicity and differentiation of cord blood (CB)-derived CD34(+) cells were investigated in serum-free medium supplemented with stem cell factor, thrombopoietin and flt3-ligand (STF). Irradiated non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were used as an animal model to assess the in vivo hematopoietic reconstitution potential of CB-derived CD34(+) cells treated by BMPs. It was demonstrated that the addition of BMP-7 at 5 ng/mL improved the proliferations of total cells, CD34(+) cells and CD34(+)CD38(-) cells without affecting the colony-forming ability of CD34(+) cells and component of lineage cells, while BMP-2 showed no effect on expanding these cells during the 10-day culture. Moreover, CB-derived CD34(+) cells cultured with STF and 5 ng/mL BMP-7 for 10 days were transplanted into irradiated NOD/SCID mice, and showed better engraftment and multi-lineage reconstitution ability compared with the cells cultured with STF alone. Together, 5 ng/mL BMP-7 was beneficial to ex vivo expansion of CB-derived CD34(+) cells for clinical purposes. The results may help improve the existing culture systems and achieve wider application of HSCs.

An investigation into the relationship between metabolic responses and energy regulation in antibody-producing cell.

Energy-efficient metabolic responses were often noted in high-productive cultures. To better understand these metabolic responses, an investigation into the relationship between metabolic responses and energy regulation was conducted via a comparative analysis among cultures with different energy source supplies. Both glycolysis and glutaminolysis were studied through the kinetic analyses of major extracellular metabolites concerning the fast and slow cell growth stages, respectively, as well as the time-course profiles of intracellular metabolites. In three cultures showing distinct antibody productivities, the amino acid metabolism and energy state were further examined. Both the transition of lactate from production to consumption and steady intracellular pools of pyruvate and lactate were observed to be correlated with efficient energy regulation. In addition, an efficient utilization of amino acids as the replenishment for the TCA cycle was also found in the cultures with upregulated energy metabolism. It was further revealed that the inefficient energy regulation would cause low cell productivity based on the comparative analysis of cell growth and productivity in cultures having distinct energy regulation.

[Impact of therapeutic time on the prognosis in critically ill patients with acute renal failure who needed renal replacement therapy].

OBJECTIVE: To investigate the impact of therapeutic time on the prognosis in critically ill patients with acute renal failure (ARF) who needed renal replacement therapy (RRT), and to analyze the risk factors of death. METHODS: All data were retrieved from the Database of Early Diagnosis and Treatment in Acute Renal Failure in Shanghai. Eighty-eight patients were collected with 56 males and 32 females who accepted continuous veno-venous hemofiltration (CVVH). The mean age was (55.73 ± 16.33) years old. Patients were divided into two groups according to therapeutic time: short time group (6-12 hours, n=49) and long time group (>12 hours, n=39). The differences between two groups before and after treatment were compared. Kaplan-Meier survival analysis, the Log-rank test was performed to evaluate the prognosis of ARF patients on 15, 30 and 60 days. Cox regression analysis was performed to evaluate the risk factors affected the patient survival. RESULTS: There were no significant difference of age, surgery, chronic kidney disease (CKD), diabetes, multiple organ failure (MOF) and severity of disease between two groups before treatment. Compared with that before treatment, blood pH, HCO(-)(3) were increased, and K(+), blood urea nitrogen (BUN), serum creatinine (SCr) were decreased after treatment (all P <0.05). There were no significant difference of Na(+) concentration and heart rate (HR), mean arterial pressure (MAP) after treatment. Kaplan-Meier survival analysis showed the survival rate of short time group and long time group were 64.4% vs. 51.4%, 52.8% vs. 46.2% and 50.4% vs.41.0% on 15, 30 and 60 days respectively. No significant difference in survival rate was noted (P=1.234). Cox regression analysis showed that the independent risk factors of short time mortality were diabetes [hazard ratio (HR)=2.134, 95% confidence interval (95%CI) 1.093-4.167,P<0.05] and MOF(HR 1.564, 95%CI 1.233-1.984,P<0.01). CONCLUSION: The mortality of ARF in critical ill patients remains high, despite accepted renal replacement therapy. The therapeutic time of CVVH may not affect the patient survival not with standing the duration of renal replacement therapy. In our group, diabetes and MOF were the independent risk factors of patients death.

Detailed understanding of enhanced specific productivity in Chinese hamster ovary cells at low culture temperature.

The specific productivity of tumor necrosis factor receptor-immunoglobulin G1 Fc fusion (TNFR-Fc) (q(TNFR-Fc)) in Chinese hamster ovary (CHO) cells at 30°C was approximately 5-fold higher than that at 37°C. To investigate reasons for increased q(TNFR-Fc) at low culture temperature, TNFR-Fc mRNA levels were determined by real-time PCR. It was found that like q(TNFR-Fc), the relative TNFR-Fc mRNA level was increased by lowering culture temperature, and more importantly, the kinetics of the increase in TNFR-Fc mRNA levels were in accordance with the changes in q(TNFR-Fc). The results demonstrated that the increased transcriptional level of TNFR-Fc was responsible for the increased q(TNFR-Fc) at low culture temperature. Enhanced levels of mRNA could derive from increased gene copy number, improved mRNA stability, or enhanced transcriptional rate. There was not a big change of gene copy number by lowering culture temperature. The transcriptional rate of TNFR-Fc was slightly decreased at 30°C, compared to 37°C. However, mRNA stability of TNFR-Fc was significantly improved by lowering culture temperature. The half-life of TNFR-Fc mRNA was 5.55 h at 30°C, whereas that was 3.69h at 37°C. Taken together, the reasons for the increased q(TNFR-Fc) in CHO cells at low culture temperature were mainly the enhanced TNFR-Fc mRNA levels, which resulted from the improved mRNA stability, rather than the changes in the gene copy number or the transcriptional rate.

Crystallization and preliminary X-ray crystallographic analysis of SMU.412c protein from the caries pathogen Streptococcus mutans.

The smu.412c gene encodes a putative histidine triad-like protein (SMU.412c) with 139 residues that is involved in cell-cycle regulation in Streptococcus mutans. The gene was cloned into the expression vector pET28a and subsequently expressed in Escherichia coli strain BL21 (DE3) to give a substantially soluble form of SMU.412c with a His(6) tag at its N-terminus. The recombinant protein was purified to homogeneity in a two-step procedure involving Ni(2+)-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained using the sitting-drop vapour-diffusion method and diffracted to 1.8 A resolution on beamline BL6A at Photon Factory, Tsukuba, Japan. The crystal belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 53.5, c = 141.1 A.