Verapamil Enhances the Activity of Caspofungin against Cryptococcus neoformans Coincides with Inhibited Ca2+ /CN Pathway and Damage with Cell Wall Integrity.

Currently, the therapeutic effect on cryptococcal infection patients is hindered by toxicity and drug resistance, making it urgent to discover alternative antifungals. Calcium channel blockers, verapamil (VER), have shown effective antifungal effects in several fungi. Here, we found that VER has a significant antifungal effect on Cryptococcus neoformans (C. neoformans). Furthermore, VER has significant synergistic effects with multiple antifungals, even caspofungin (CAS). We confirmed that VER and CAS had a synergistic antifungal effect in the Galleria mellonella. We conducted in-depth research on the possible mechanism of the synergistic impact of VER and CAS. After treatment with VER, the chitosan content of C. neoformans ' cell wall decreased and in dopamine liquid culture medium, we observed the leakage of melanin. Through cell wall fluorescence staining and stress sensitivity analysis, we further demonstrated that VER impaired the integrity of the C. neoformans' cell wall. Another side, VER+CAS modification of C. neoformans membrane permeability, leading to intracellular ROS accumulation and mitochondrial membrane potential changes. VER eliminated the cytoplasmic calcium fluctuations caused by CAS stimulation and down-regulated the genes expression associated with the calcineurin pathway. In addition, we found that the enzyme activity of chitin deacetylase of C. neoformans is significantly influenced by the presence of Ca2+, suggesting that the use of VER may affect the activity. In summary, the synergistic antifungal effect of VER and CAS makes them effective and promising candidates for fungal treatment.

Pilonidal cyst of the scalp.

A 19-year-old male patient presented with a slow-growing mass on his scalp over a 10-year history. After resecting the mass, he was diagnosed with pilonidal cyst based on the clinical and histological findings. An unusual pilonidal disease is observed on the scalp in this report.

Simplified and effective RNA interference and CRISPR-Cas9 systems for Cryptococcus neoformans.

The 3,4-dihydroxyphenylalanine (DOPA) melanin is one of the important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. While the production of DOPA melanin is catalyzed by laccase that is predominantly encoded by LAC1 gene. Therefore, regulating the genetic expression of C. neoformans is conducive to exploring the impact of interested molecules on the host. In this work, we established two systems that were constructed quickly and easily for the knock-down/knock-out of LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats CRISPR-Cas9. The RNAi system was constructed by pSilencer 4.1-CMV neo plasmid and short hairpin RNA to achieve effective transcriptional suppression. The CRISPR-Cas9 system was used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, quantitative real-time polymerase chain reaction, transmission electron microscope, and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA was more powerful and lasted longer. An albino strain produced by CRISPR-Cas9 was completely unable to synthesize melanin. In conclusion, strains with different capacities of melanin production were obtained by RNAi and CRISPR-Cas9 systems, which might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, the two systems in this article might be convenient to quickly screen the possible trait-regulating genes of other serotypes of C. neoformans.

Effect and mechanism of hydrogen-rich bath on mice with imiquimod-induced psoriasis.

The purpose of this study was to investigate whether hydrogen-rich bath has therapeutic effect on psoriasis and its molecular mechanism. Mice with imiquimod-induced psoriasis were established and divided into groups. The mice were respectively treated with hydrogen-rich water bath and distilled water bath. The changes of skin lesions and PSI scores of mice were compared after their treatments. HE staining was used to observe the pathological feature. The changes of inflammatory indexes and immune factors were analysed by ELISA and immunohistochemical staining. Malondialdehyde (MDA) content was measured by the thiobarbituric assay (TBA) method. By naked eye, the severity of skin lesions in hydrogen-rich water bath group was lower than that in distilled water bath group, and the psoriasis severity index (PSI) was lower (p < 0.01). The results of HE staining showed that the mice with distilled water bath had more abnormal keratosis, thickening of the spinous layer and prolongation of the dermal process, and more Munro abscess than the mice with hydrogen-rich water bath. During the course of disease, the overall levels and peaks of IL-17, IL-23, TNF-α, CD3+ and MDA in mice with hydrogen-rich bath were lower than those in mice with distilled water bath (p < 0.05). In the skin, the mice treated with the hydrogen-rich water bath also had lower peak of proliferating cell nuclear antigen (PCNA) levels. It is concluded that hydrogen-rich water bath can inhibit psoriasis inflammation and oxidative stress, relieve psoriasis skin lesions and accelerate the end of abnormal skin proliferation state, which shows a therapeutic and improving effect on psoriasis.

Recalcitrant Palmar-Plantar Atopic Dermatitis Successfully Treated with Dupilumab: A Case Series.

Background: Palmar-plantar atopic dermatitis is a common and debilitating condition with significant diagnostic and management challenges. Case Presentation: The paper presents the successful treatment of dupilumab in five female patients with recalcitrant palmar-plantar atopic dermatitis. Methods: The Hand Eczema Severity Index (HECSI) and the Foot Eczema Severity Index (FECSI), eosinophils and IgE levels were evaluated before and after the treatment. Results: Five recalcitrant palmar-plantar atopic dermatitis patients had substantial responses to dupilumab and the persistent pruritus was soon alleviated during the therapy. By week 16, all patients' palmar-plantar eczema has been almost clear, with significant decrease of HECSI and FECSI. The count of eosinophils and IgE levels returned to normal in all patients. Conclusion: Dupilumab significantly improves disease severity and reduces eosinophils and IgE in refractory palmar-plantar AD patients.

VDAC1 promotes cardiomyocyte autophagy in anoxia/reoxygenation injury via the PINK1/Parkin pathway.

Ischemia/reperfusion (I/R) is a well-known injury to the myocardium, but the mechanism involved remains elusive. In addition to the well-accepted apoptosis theory, autophagy was recently found to be involved in the process, exerting a dual role as protection in ischemia and detriment in reperfusion. Activation of autophagy is mediated by mitochondrial permeability transition pore (MPTP) opening during reperfusion. In our previous study, we showed that MPTP opening is regulated by VDAC1, a channel protein located in the outer membrane of mitochondria. Thus, upregulation of VDAC1 expression is a possible trigger to cardiomyocyte autophagy via an unclear pathway. Here, we established an anoxia/reoxygenation (A/R) model in vitro to simulate the I/R process in vivo. At the end of A/R treatment, VDAC1, Beclin 1, and LC3-II/I were upregulated, and autophagic vacuoles were increased in cardiomyocytes, which showed a connection of VDAC1 and autophagy development. These variations also led to ROS burst, mitochondrial dysfunction, and aggravated apoptosis. Knockdown of VDAC1 by RNAi could alleviate the above-mentioned cellular damages. Additionally, the expression of PINK1 and Parkin was enhanced after A/R injury. Furthermore, Parkin was recruited to mitochondria from the cytosol, which suggested that the PINK1/Parkin autophagic pathway was activated during A/R. Nevertheless, the PINK1/Parkin pathway was effectively inhibited when VDAC1 was knocked-down. Taken together, the A/R-induced cardiomyocyte injury was mediated by VDAC1 upregulation, which led to cell autophagy via the PINK1/Parkin pathway, and finally aggravated apoptosis.