Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine (CQ) has played an indispensable role, however, its mechanism of action (MoA) is not fully understood.

Methods: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling (ABPP) and mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays.

Results: We developed a novel clickable, photo-affinity chloroquine analog probe (CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photocrosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods.

Conclusions: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled (ABPP) and label-free (MS-CETSA) methods.

Background: Sepsis involves life-threatening organ dysfunction and is caused by a dysregulated host response to infection. No specific therapies against sepsis have been reported. Celastrol (Cel) is a natural anti-inflammatory compound that shows potential against systemic inflammatory diseases. This study aimed to investigate the pharmacological activity and molecular mechanism of Cel in models of endotoxemia and sepsis.

Methods: We evaluated the anti-inflammatory efficacy of Cel against endotoxemia and sepsis in mice and macrophage cultures treated with lipopolysaccharide (LPS). We screened for potential protein targets of Cel using activity-based protein profiling (ABPP). Potential targets were validated using biophysical methods such as cellular thermal shift assays (CETSA) and surface plasmon resonance (SPR). Residues involved in Cel binding to target proteins were identified through point mutagenesis, and the functional effects of such binding were explored through gene knockdown.

Results: Cel protected mice from lethal endotoxemia and improved their survival with sepsis, and it significantly decreased the levels of pro-inflammatory cytokines in mice and macrophages treated with LPS (P<0.05). Cel bound to Cys424 of pyruvate kinase M2 (PKM2), inhibiting the enzyme and thereby suppressing aerobic glycolysis (Warburg effect). Cel also bound to Cys106 in high mobility group box 1 (HMGB1) protein, reducing the secretion of inflammatory cytokine interleukin (IL)-1β. Cel bound to the Cys residues in lactate dehydrogenase A (LDHA).

Conclusions: Cel inhibits inflammation and the Warburg effect in sepsis via targeting PKM2 and HMGB1 protein.

Background: Mucosal-associated invariant T (MAIT) cells are systemically depleted in human immunodeficiency virus type 1 (HIV-1) infected patients and are not replenished even after successful combined antiretroviral therapy (cART). This study aimed to identify the mechanism underlying MAIT cell depletion.

Methods: In the present study, we applied flow cytometry, single-cell RNA sequencing and immunohistochemical staining to evaluate the characteristics of pyroptotic MAIT cells in a total of 127 HIV-1 infected individuals, including 69 treatment-naive patients, 28 complete responders, 15 immunological non-responders, and 15 elite controllers, at the Fifth Medical Center of Chinese PLA General Hospital, Beijing, China.

Results: Single-cell transcriptomic profiles revealed that circulating MAIT cells from HIV-1 infected subjects were highly activated, with upregulation of pyroptosis-related genes. Further analysis revealed that increased frequencies of pyroptotic MAIT cells correlated with markers of systemic T-cell activation, microbial translocation, and intestinal damage in cART-naive patients and poor CD4⁺ T-cell recovery in long-term cART patients. Immunohistochemical staining revealed that MAIT cells in the gut mucosa of HIV-1 infected patients exhibited a strong active gasdermin-D (GSDMD, marker of pyroptosis) signal near the cavity side, suggesting that these MAIT cells underwent active pyroptosis in the colorectal mucosa. Increased levels of the proinflammatory cytokines interleukin-12 (IL-12) and IL-18 were observed in HIV-1 infected patients. In addition, activated MAIT cells exhibited an increased pyroptotic phenotype after being triggered by HIV-1 virions, T-cell receptor signals, IL-12 plus IL-18, and combinations of these factors, in vitro.

Conclusions: Activation-induced MAIT cell pyroptosis contributes to the loss of MAIT cells in HIV-1 infected patients, which could potentiate disease progression and poor immune reconstitution.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor, and it is associated with poor prognosis. Its characteristics of being highly invasive and undergoing heterogeneous genetic mutation, as well as the presence of the blood–brain barrier (BBB), have reduced the efficacy of GBM treatment. The emergence of a novel therapeutic method, namely, sonodynamic therapy (SDT), provides a promising strategy for eradicating tumors via activated sonosensitizers coupled with low-intensity ultrasound. SDT can provide tumor killing effects for deep-seated tumors, such as brain tumors. However, conventional sonosensitizers cannot effectively reach the tumor region and kill additional tumor cells, especially brain tumor cells. Efforts should be made to develop a method to help therapeutic agents pass through the BBB and accumulate in brain tumors. With the development of novel multifunctional nanosensitizers and newly emerging combination strategies, the killing ability and selectivity of SDT have greatly improved and are accompanied with fewer side effects. In this review, we systematically summarize the findings of previous studies on SDT for GBM, with a focus on recent developments and promising directions for future research.

The literature is full of claims regarding the consumption of polyphenol or polyamine-rich foods that offer some protection from developing cardiovascular disease (CVD). This is achieved by preventing cardiac hypertrophy and protecting blood vessels through improving the function of endothelium. However, do these interventions work in the aged human hearts? Cardiac aging is accompanied by an increase in left ventricular hypertrophy, along with diastolic and systolic dysfunction. It also confers significant cardiovascular risks for both sexes. The incidence and prevalence of CVD increase sharply at an earlier age in men than women. Furthermore, the patterns of heart failure differ between sexes, as do the lifetime risk factors. Do caloric restriction (CR)-mimetics, rich in polyphenol or polyamine, delay or reverse cardiac aging equally in both men and women? This review will discuss three areas: (1) mechanisms underlying age-related cardiac remodeling; (2) gender-related differences and potential mechanisms underlying diminished cardiac response in older men and women; (3) we select a few polyphenol or polyamine rich compounds as the CR-mimetics, such as resveratrol, quercetin, curcumin, epigallocatechin gallate and spermidine, due to their capability to extend health-span and induce autophagy. We outline their abilities and issues on retarding aging in animal hearts and preventing CVD in humans. We discuss the confounding factors that should be considered for developing therapeutic strategies against cardiac aging in humans.

Military psychiatry, a new subcategory of psychiatry, has become an invaluable, intangible effect of the war. In this review, we begin by examining related military research, summarizing the related epidemiological data, neuropathology, and the research achievements of diagnosis and treatment technology, and discussing its comorbidity and sequelae. To date, advances in neuroimaging and molecular biology have greatly boosted the studies on military traumatic brain injury (TBI). In particular, in terms of pathophysiological mechanisms, several preclinical studies have identified abnormal protein accumulation, blood–brain barrier damage, and brain metabolism abnormalities involved in the development of TBI. As an important concept in the field of psychiatry, TBI is based on organic injury, which is largely different from many other mental disorders. Therefore, military TBI is both neuropathic and psychopathic, and is an emerging challenge at the intersection of neurology and psychiatry.

Background: Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive.

Methods: The 150 male C57 mice underwent middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h, Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1 (Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine (NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA (mtDNA) copy number, intracellular and mitochondrial reactive oxygen species (ROS), autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/I, TNF-α, IL-1β, etc., were detected under normal or Drp1 interference conditions.

Results: The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI (P<0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44% and 88% by Drp1 short hairpin RNA (shRNA) (P<0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC (P<0.05). RIP1/RIP3 inhibitor Necrostatin-1 (Nec-1) restored 75% to a low LC3 II/I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation (P<0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes (P>0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4–5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1 (P<0.05). Furthermore, TNF-α and IL-1β increased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen–glucose deprivation/reoxygenation (OGD/R) conditions (P<0.05).

Conclusions: CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation, triggering a vicious cycle.

Background: Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands (SGs), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin.

Methods: The expression of the SG markers cytokeratin 5 (CK5), cytokeratin 10 (CK10), cytokeratin 18 (CK18), carcino-embryonic antigen (CEA), aquaporin 5 (AQP5) and α-smooth muscle actin (α-SMA) was assessed with quantitative PCR (qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells (iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs. BALB/c nude mice were randomly divided into normal group, SGM treatment group and iSGC transplantation group. Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs.

Results: Ectodermal dysplasia antigen (EDA) overexpression drove human dermal fibroblast (HDF) conversion into iSGCs in SG culture medium (SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18 and CEA in iSGCs, and flow cytometry data demonstrated (4.18±0.04)% of iSGCs were CK5 positive and (4.36±0.25)% of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails, (23.05±2.49)% of iSGCs expressed CK5⁺ and (55.79±3.18)% of iSGCs expressed CK18⁺, respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79±7.71)% vs. (70.59±0.34)%, ns]. In vivo transplantation experiments showed approximately (5.2±1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice.

Conclusions: We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.

Background: Airway inflammation is the core pathological process of asthma, with the key inflammatory regulators incompletely defined. Recently, fibroblast growth factor 2 (FGF2) has been reported to be an inflammatory regulator; however, its role in asthma remains elusive. This study aimed to investigate the immunomodulatory role of FGF2 in asthma.

Methods: First, FGF2 expression was characterised in clinical asthma samples and the house dust mite (HDM)-induced mouse chronic asthma model. Second, recombinant mouse FGF2 (rm-FGF2) protein was intranasally delivered to determine the effect of FGF2 on airway inflammatory cell infiltration. Third, human airway epithelium-derived A549 cells were stimulated with either HDM or recombinant human interleukin-1β (IL-1β) protein combined with or without recombinant human FGF2. IL-1β-induced IL-6 or IL-8 release levels were determined using enzyme-linked immunosorbent assay, and the involved signalling transduction was explored via Western blotting.

Results: Compared with the control groups, the FGF2 protein levels were significantly upregulated in the bronchial epithelium and alveolar areas of clinical asthma samples [(6.70±1.79) vs. (16.32±2.40), P=0.0184; (11.20±2.11) vs. (21.00±3.00), P=0.033, respectively] and HDM-induced asthmatic mouse lung lysates [(1.00±0.15) vs. (5.14±0.42), P<0.001]. Moreover, FGF2 protein abundance was positively correlated with serum total and anti-HDM IgE levels in the HDM-induced chronic asthma model (R²=0.857 and 0.783, P=0.0008 and 0.0043, respectively). Elevated FGF2 protein was mainly expressed in asthmatic bronchial epithelium and alveolar areas and partly co-localised with infiltrated inflammatory cell populations in HDM-induced asthmatic mice. More importantly, intranasal instillation of rm-FGF2 aggravated airway inflammatory cell infiltration [(2.45±0.09) vs. (2.88±0.14), P=0.0288] and recruited more subepithelial neutrophils after HDM challenge [(110.20±29.43) cells/mm² vs. (238.10±42.77) cells/mm², P=0.0392] without affecting serum IgE levels and Th2 cytokine transcription. In A549 cells, FGF2 was upregulated through HDM stimulation and promoted IL-1β-induced IL-6 or IL-8 release levels [up to (1.41±0.12)- or (1.44±0.14)-fold change vs. IL-1β alone groups, P=0.001 or 0.0344, respectively]. The pro-inflammatory effect of FGF2 is likely mediated through the fibroblast growth factor receptor (FGFR)/mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB) pathway.

Conclusions: Our findings suggest that FGF2 is a potential inflammatory modulator in asthma, which can be induced by HDM and acts through the FGFR/MAPK/NF-κB pathway in the airway epithelial cells.

Novel advances in the field of brain imaging have enabled the unprecedented clinical application of various imaging modalities to facilitate disease diagnosis and treatment. Electrical impedance tomography (EIT) is a functional imaging technique that measures the transfer impedances between electrodes on the body surface to estimate the spatial distribution of electrical properties of tissues. EIT offers many advantages over other neuroimaging technologies, which has led to its potential clinical use. This qualitative review provides an overview of the basic principles, algorithms, and system composition of EIT. Recent advances in the field of EIT are discussed in the context of epilepsy, stroke, brain injuries and edema, and other brain diseases. Further, we summarize factors limiting the development of brain EIT and highlight prospects for the field. In epilepsy imaging, there have been advances in EIT imaging depth, from cortical to subcortical regions. In stroke research, a bedside EIT stroke monitoring system has been developed for clinical practice, and data support the role of EIT in multi-modal imaging for diagnosing stroke. Additionally, EIT has been applied to monitor the changes in brain water content associated with cerebral edema, enabling the early identification of brain edema and the evaluation of mannitol dehydration. However, anatomically realistic geometry, inhomogeneity, cranium completeness, anisotropy and skull type, etc., must be considered to improve the accuracy of EIT modeling. Thus, the further establishment of EIT as a mature and routine diagnostic technique will necessitate the accumulation of more supporting evidence.