Core N-Glycan Structures Are Critical for the Pathogenicity of Cryptococcus neoformans by Modulating Host Cell Death.

Cryptococcus neoformans is a human-pathogenic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised individuals. To investigate the roles of N-glycan core structure in cryptococcal pathogenicity, we constructed mutant strains of C. neoformans with defects in the assembly of lipid-linked N-glycans in the luminal side of the endoplasmic reticulum (ER). Deletion of ALG3 (alg3Δ), which encodes dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3-mannosyltransferase, resulted in the production of truncated neutral N-glycans carrying five mannose residues as a major species. Despite moderate or nondetectable defects in virulence-associated phenotypes in vitro, the alg3Δ mutant was avirulent in a mouse model of systemic cryptococcosis. Notably, the mutant did not show defects in early stages of host cell interaction during infection, including attachment to lung epithelial cells, opsonic/nonopsonic phagocytosis, and manipulation of phagosome acidification. However, the ability to drive macrophage cell death was greatly decreased in this mutant, without loss of cell wall remodeling capacity. Furthermore, deletion of ALG9 and ALG12, encoding Dol-P-Man-dependent α-1,2-mannosyltransferases and α-1,6-mannosyltransferases, generating truncated core N-glycans with six and seven mannose residues, respectively, also displayed remarkably reduced macrophage cell death and in vivo virulence. However, secretion levels of interleukin-1ß (IL-1ß) were not reduced in the bone marrow-derived dendritic cells obtained from Asc- and Gsdmd-deficient mice infected with the alg3Δ mutant strain, excluding the possibility that pyroptosis is a main host cell death pathway dependent on intact core N-glycans. Our results demonstrated N-glycan structures as a critical feature in modulating death of host cells, which is exploited by as a strategy for host cell escape for dissemination of C. neoformansIMPORTANCE We previously reported that the outer mannose chains of N-glycans are dispensable for the virulence of C. neoformans, which is in stark contrast to findings for the other human-pathogenic yeast, Candida albicans Here, we present evidence that an intact core N-glycan structure is required for C. neoformans pathogenicity by systematically analyzing alg3Δ, alg9Δ, and alg12Δ strains that have defects in lipid-linked N-glycan assembly and in in vivo virulence. The alg null mutants producing truncated core N-glycans were defective in inducing host cell death after phagocytosis, which is triggered as a mechanism of pulmonary escape and dissemination of C. neoformans, thus becoming inactive in causing fatal infection. The results clearly demonstrated the critical features of the N-glycan structure in mediating the interaction with host cells during fungal infection. The delineation of the roles of protein glycosylation in fungal pathogenesis not only provides insight into the glycan-based fungal infection mechanism but also will aid in the development of novel antifungal agents.

The effect of palonosetron on rocuronium-induced withdrawal movement / O efeito de palonosetron sobre o movimento de retração induzido por rocurônio

Abstract Background: Rocuronium causes pain and withdrawal movement during induction of anesthesia. In this study, palonosetron was investigated to have analgesic effect on the reduction of rocuronium-induced withdrawal movement. Methods: 120 patients were randomly assigned to one of three groups to receive either saline, lidocaine 20 mg, or palonosetron 0.075 mg with a tourniquet applied two minutes before thiopental sodium (5 mg.kg-1) was given intravenously. After loss of consciousness, rocuronium (0.6 mg.kg-1) was injected and the withdrawal movement was estimated by 4-point scale in a double-blind manner. Results: The overall incidence of rocuronium withdrawal movement was 50% with lidocaine (p = 0.038), 38% with palonosetron (p = 0.006) compared with 75% for saline. The incidence of no pain to mild pain was significantly lower in the lidocaine and palonosetron groups (85% and 92% respectively) than in the saline group (58%). However, there was no significant difference in withdrawal movement between the lidocaine and palonosetron groups. There was no severe movement with palonosetron. Conclusion: Pretreatment of palonosetron with venous occlusion may attenuate rocuronium-induced withdrawal movement as effective as the use of lidocaine. It suggested that peripheral action of palonosetron was effective to reduce rocuronium-induced withdrawal movement.

Resumo Justificativa: Rocurônio provoca dor e reflexo de retirada durante a indução da anestesia. Neste estudo, avaliamos se palonosetron tem efeito analgésico para reduzir esse movimento induzido por rocurónio. Métodos: Cento e vinte pacientes foram randomicamente designados para um de três grupos para receber solução salina, lidocaína (20 mg) ou palonosetron (0.075 mg), com aplicação de torniquete dois minutos antes da administração intravenosa de tiopental sódico (5 mg.kg-1). Após a perda de consciência, rocurônio (0.6 mg.kg-1) foi injetado e o reflexo de retirada foi avaliado com o uso de uma escala de quatro pontos, de modo duplo-cego. Resultados: A incidência global do reflexo de retirada induzido por rocurônio foi de 50% para lidocaína (p = 0,038), 38% para palonosetron (p = 0,006), em comparação com 75% para solução salina. A incidência de dor ausente ou leve foi significativamente menor nos grupos lidocaína e palonosetron (85% e 92%, respectivamente) que no grupo solução salina (58%). Porém, não houve diferença significativa no reflexo de retirada entre os grupos lidocaína e palonosetron. Não houve movimento grave com palonosetron. Conclusão: O pré-tratamento com palonosetron com oclusão venosa pode atenuar o reflexo de retirada induzido por rocurônio de modo tão eficaz como o uso de lidocaína. Sugeriu-se que a ação periférica de palonosetron foi eficaz para reduzir o reflexo de retirada induzido por rocurônio.

Treatment of radiation-induced cystitis and vulvodynia via a ganglion impar block using a lateral approach under computed tomography guidance: a case report.

Adjuvant radiation therapy (RT) after colorectal cancer surgery can prevent local recurrence, but has several side effects. Precise injection of drugs into the affected areas is complicated by radiation-induced fibrosis of soft or connective tissue. A 48-year-old woman experienced severe intractable perineal pain, dysuria, urinary urgency, and frequent urination after rectal cancer surgery and adjuvant RT, and was diagnosed with radiation-induced cystitis and vulvodynia. Her symptoms persisted despite two fluoroscopy-guided ganglion impar blocks. Fluoroscopy revealed atypical needle tip positioning and radiolucent dye distribution, presumably due to radiation-induced fibrosis in the target region. We performed two computed tomography (CT)-guided ganglion impar blocks by using a lateral approach, which allowed more accurate po-sitioning of the needle tip. Her pain visual analog score decreased from 9 to 3, and she recently resumed sexual intimacy. CT guidance is a viable alternative to fluoroscopy guidance when performing ganglion impar blocks in fibrotic areas.

Use of the cysteine-repressible HpMET3 promoter as a novel tool to regulate gene expression in Hansenula polymorpha.

OBJECTIVES: The promoter of HpMET3, encoding an ATP sulfurylase, was evaluated for its potential as a repressible promoter to downregulate the expression of target genes in the thermotolerant, methylotrophic yeast Hansenula polymorpha. RESULTS: The expression of lacZ under the control of the 0.6 kb HpMET3 promoter was efficiently downregulated by cysteine, but not by methionine or sulfate. The HpMET3 promoter was used to generate a conditional mutant of the HpPMT2 gene encoding an O-mannosyltransferase, which is involved in post-translational protein modification. The addition of 0.5 mM cysteine adversely affected the growth of the conditional HpMET3(p)-Hppmt2 mutant strain by downregulating transcription of HpPMT2 to approx. 40 % of the normal levels, indicating that the HpPMT2 gene is essential for cell viability. However, the HpMET3 promoter was neither induced nor repressed in the heterologous host Saccharomyces cerevisiae. CONCLUSION: Our results reveal that the cysteine-repressible HpMET3 promoter is a useful tool that downregulates the expression of various genes in H. polymorpha.