Rapid signal transduction in living cells is a unique feature of mechanotransduction

It is widely postulated that mechanotransduction is initiated at the local force-membrane interface by inducing local conformational changes of proteins, similar to soluble ligand-induced signal transduction. However, all published reports are limited in time scale to address this fundamental issue. Using a FRET-based cytosolic Src reporter in a living cell, we quantified changes of Src activities as a local stress via activated integrins was applied. The stress induced rapid (<0.3 s) activation of Src at remote cytoplasmic sites, which depends on the cytoskeletal prestress. In contrast, there was no Src activation within 12 s of soluble epidermal growth factor (EGF) stimulation. A 1.8-Pa stress over a focal adhesion activated Src to the same extent as 0.4 ng/ml EGF at long times (minutes), and the energy levels for mechanical stimulation and chemical stimulation were comparable. The effect of both stress and EGF was less than additive. Nanometer-scale cytoskeletal deformation analyses revealed that the strong activation sites of Src by stress colocalized with large deformation sites of microtubules, suggesting that microtubules are essential structures for transmitting stresses to activate cytoplasmic proteins. These results demonstrate that rapid signal transduction via the prestressed cytoskeleton is a unique feature of mechanotransduction.     

Caldesmon-dependent switching between capillary endothelial cell growth and apoptosis through modulation of cell shape and contractility

Caldesmon (CaD), a protein component of the actomyosin filament apparatus, modulates cell shape and cytoskeletal structure when overexpressed. When capillary endothelial cells were infected with an adenoviral vector encoding GFP-CaD under Tet-Off control, progressive inhibition of contractility, loss of actin stress fibers, disassembly of focal adhesions, and cell retraction resulted. This was accompanied by a cell shape (rounding)-dependent increase in apoptosis and concomitant inhibition of cell cycle progression. Cell growth also was inhibited in low expressor cells in which cell tension was suppressed independently of significant changes in cell shape, cytoskeletal structure, or focal adhesions. Thus, changes in both cytoskeletal structure and contractility appear to be central to the mechanism by which extracellular matrix-dependent changes in capillary cell shape influence growth and apoptosis during angiogenesis, and hence the cytoskeleton may represent a potential target for anti-angiogenesis therapy.     

Cytokines and fungal infections

The very poor outcome of invasive fungal infections (IFI) in patients with haematological malignancies or recipients of haematopoietic stem cell transplantation is largely attributed to their compromised host defence mechanisms. The restoration or augmentation of immune responses in these patients is now considered as one of the cornerstones of effective antifungal therapy. Major advances in the field of experimental immunology have provided insight on the important regulatory role of cytokines in both innate and adaptive immunity to fungal pathogens. Preclinical studies have convincingly demonstrated that immunomodulation with cytokines can enhance the antifungal activity of neutrophils and monocytes/macrophages as well as upregulate protective T-helper type 1 adaptive immune responses. Evidence on the clinical use of cytokines in immunocompromised hosts with IFI is, however, still scant and inconclusive. The present review summarizes experimental and clinical data on the role of cytokines in the immune response to fungal pathogens and on their potential use for prevention or treatment of fungal infections. Implications for future research are also briefly discussed.     

Dandruff-associated Malassezia genomes reveal convergent and divergent virulence traits shared with plant and human fungal pathogens

Fungi in the genus Malassezia are ubiquitous skin residents of humans and other warm-blooded animals. Malassezia are involved in disorders including dandruff and seborrheic dermatitis, which together affect >50% of humans. Despite the importance of Malassezia in common skin diseases, remarkably little is known at the molecular level. We describe the genome, secretory proteome, and expression of selected genes of Malassezia globosa. Further, we report a comparative survey of the genome and secretory proteome of Malassezia restricta, a close relative implicated in similar skin disorders. Adaptation to the skin environment and associated pathogenicity may be due to unique metabolic limitations and capabilities. For example, the lipid dependence of M. globosa can be explained by the apparent absence of a fatty acid synthase gene. The inability to synthesize fatty acids may be complemented by the presence of multiple secreted lipases to aid in harvesting host lipids. In addition, an abundance of genes encoding secreted hydrolases (e.g., lipases, phospholipases, aspartyl proteases, and acid sphingomyelinases) was found in the M. globosa genome. In contrast, the phylogenetically closely related plant pathogen Ustilago maydis encodes a different arsenal of extracellular hydrolases with more copies of glycosyl hydrolase genes. M. globosa shares a similar arsenal of extracellular hydrolases with the phylogenetically distant human pathogen, Candida albicans, which occupies a similar niche, indicating the importance of host-specific adaptation. The M. globosa genome sequence also revealed the presence of mating-type genes, providing an indication that Malassezia may be capable of sex.     

辽吉两省部分鹿场鹿茸皮肤病的临床特征与真菌分离初探

目的 研究探讨生茸期梅花鹿、马鹿在鹿茸快速生长期常发生以“白皮茸”、“痂皮茸”为主要症状的皮肤病。方法 研究对吉林、辽宁8个梅花鹿、马鹿养殖场的20份患病鹿进行了临床观察、血细胞计数、病理观察、病原分离及ITS分子鉴定。结果 发现本病仅侵染鹿茸皮肤表层、主要病变为结痂处真皮组织坏死、炎性细胞浸润; 血液中淋巴细胞、白细胞和中性粒细胞数变化不明显。对茸皮患处分离菌的鉴定结果表明68株分离株中44株属于半知菌亚门(64.7%), 24株属于酵母亚门(35.3%), 其中, 半知菌亚门的毛癣菌属、表皮癣菌属以及酵母亚门的念珠菌等可能为本病的条件致病菌。结论 鹿茸“白皮茸”、“痂皮茸”病均符合真菌皮肤病的发病特征,为该类疾病的预防及治疗提供了一定的参考。     

侵袭性真菌早期诊断和治疗的研究进展

近十多年来，随着免疫缺陷患者数量的增多，广谱抗菌素的广泛使用等因素的出现，侵袭性真菌感染（IFI）日益增多。目前IFI已经成为各器官深部真菌最严重感染之一，勿庸质疑早期诊断和及时治疗会大大提高患者生存率。在近几年里，真菌抗原和基因组DNA检测技术的更新是诊断方面的进步，而一些抗真菌药物也有了发展，如两性霉素B的脂类抑制剂、新一代咪唑类和棘球白素类等。本文就其早期诊断和治疗的进展这两大方面进行了综述。     

肺部真菌感染的诊治进展

近些年来对肺部真菌感染的认识取得了较大的进展,除了传统的支气管镜检查外,新的诊断方法还有免疫学和分子生物学检测,治疗方面,新型抗真菌药物不断开发。但因其临床表现特异性小,病死率高,早期诊断和治疗仍存在困难,发病呈上升趋势,应引起临床高度重视。     

肺结核抗痨治疗与真菌培养阳性相关性分析

目的探讨肺结核患者与痰真菌培养阳性之间的相关性。方法对我院收治的126例患者抗结核治疗过程中痰真菌培养阳性出现时间、相关因素及预后进行分析。结果抗结核药物联合应用糖皮质激素的患者,痰真菌培养阳性出现时间早于单纯抗结核药物治疗的患者;我院痰液真菌培养生长菌株种类主要为念珠菌和丝状菌;有临床症状的患者抗真菌联合免疫增强剂应用后痰转阴率与无临床症状的患者仅用免疫增强剂后转阴率差别无统计学意义。结论 1.糖皮质激素联合抗结核药物有加快呼吸系统菌群失调的可能;2.结核病患者治疗过程中出现无症状的痰真菌培养阳性患者可不予抗真菌治疗。     

Invasive fungal infection following venetoclax and posaconazole co-administration

The co-administration of venetoclax, a BCL-2 inhibitor, with a mould-active azole, such a posaconazole, has potential to both prevent invasive fungal infection (IFI) and reduce the required treatment dose, and cost, of venetoclax. Posaconazole drug-level monitoring is critical to ensuring adequate mould prophylaxis. A retrospective audit of 99 patients at a tertiary cancer centre, with myeloid malignancies co-prescribed venetoclax and posaconazole between January 2018 and April 2022, was undertaken to evaluate the adequacy of posaconazole prescribing and the rate of breakthrough IFI. Seventy-six patients (77%) had at least one posaconazole level measured in the study period, with 37% requiring a dose adjustment based on steady-state trough levels. Breakthrough IFI occurred in 4% of patients, typically within 1 month of commencing anti-mould prophylaxis. Close monitoring of posaconazole levels in venetoclax-treated patients, particularly in the early, outpatient setting, is critical.     

Two-step process of cytoskeletal structural damage during long-term storage of packed red blood cells

BackgroundStorage of packed red blood cells (PRBC) for 42 days causes morphological, structural, and functional changes in the red cells. To assess the quality of stored PRBC, it is important to evaluate the main components of the product. The aim of this study was to evaluate the kinetics of the structural transformations in the cytoskeleton of red cells during long-term storage (up to 42 days).Materials and methodsBags of PRBC were stored with CPD/SAGM solution at +4 °C. Cytoskeletal parameters were measured on days 3, 12, 19, 21, 24, 28, 35, and 42 of storage to determine their changes. Atomic force microscopy was used to obtain images and analyse the parameters of the cytoskeletal network. As the storage time increased, a general PRBC test was performed. Membrane fixatives were not used at any stage of the preparation of the specimens for cytoskeletal imaging.ResultsWhen PRBC were stored for 42 days, the main changes to the cytoskeletal mesh included rupture of filaments, merger of small pores into larger ones, a decrease of the number of pores, thickening of filaments, and an increase of membrane stiffness. A process of irreversible changes to the cytoskeleton started on days 19–21. A kinetic model of changes in the parameters of the cytoskeletal mesh with time of PRBC storage was created.DiscussionTwo stages of impairment in cytoskeletal elements were found: rupture of filaments and clustering of protein components. The typical time of development and specifics of these stages are discussed. The consequences of the altered configuration of the cytoskeleton are also discussed. Destruction of the red cell cytoskeleton can have a negative effect on the efficacy of blood transfusion and increase the risk of post-transfusion complications. Our findings can be used in clinical medicine to evaluate the quality of PRBC for blood transfusion as well as for studies of the molecular organisation of red cells undergoing various types of physical and chemical treatment.     

Prevalence and outcome of fungal infection in patients with severe acute pancreatitis

Background and Aim:  To study the prevalence of risk factors and outcome of fungal infections in patients with severe acute pancreatitis.
Methods:  Fifty consecutive patients with severe acute pancreatitis were investigated for evidence of fungal infection by weekly culture of body fluids and aspirate from pancreatic/peripancreatic tissue and samples collected at necrosectomy. All patients were managed as per a standard protocol. Patients with documented fungal infection were treated with intravenous amphotericin or fluconazole. Data were analyzed using SPSS software (version 13), and risk factors for fungal infection and mortality were determined.
Results:  Fungal infection was documented in 18 (36%) of 50 patients with Candida albicans (the commonest species). The incidence of fungal infection steadily increased with increasing duration of hospital stay. Those with fungal infection more often had evidence of respiratory failure ( P  = 0.031) and hypotension ( P  = 0.031) at admission, prolonged hospital stay > 4 weeks ( P  = 0.034), longer duration of antibiotics ( P  = 0.003), received total parenteral nutrition ( P  = 0.005), and required mechanical ventilation ( P  = 0.001) in contrast to those without fungal infection. The logistic regression analysis found the independent risk factors for fungal infection to be antibiotic therapy for > 4 weeks and hypotension at hospitalization. Of the 18 patients with fungal infection, 13 were administered intravenous antifungals; eight of these patients survived, while the five who did not receive antifungals died.
Conclusion:  Fungal infection was detected in 36% of our patients. The independent risk factors associated with it were hypotension at hospitalization and prolonged antibiotic therapy. Antifungal therapy improved their chances of survival.     

肺真菌病59例病原学和影像学分析

目的：探讨肺真菌病的病原学分布和影像学特征。方法收集肺真菌病59例,均经支气管镜、经皮肺穿刺活检或手术切除送病理学确诊,分析其病原学分布和影像学特征。结果59例病理学确诊肺真菌病患者中,肺曲霉病24例（40．7％）,肺隐球菌病24例（40．7％）,肺毛霉病5例（8．5％）,肺念珠菌病4例（6．8％）,组织胞浆菌病2例（3．4％）,合并放线菌肺炎1例（1．7％）。胸部影像学改变包括肺部肿块23例（39．0％）,渗出性病变23例（39．0％）,结节8例（13．6％）,支气管肿物3例（5．1％）,空洞病变1例（1．7％）,弥漫性病变1例（1．7％）。误诊为肺炎12例（20．3％）,肺结核7例（11．9％）,肺癌4例（6．8％）。结论病理学确诊的肺真菌病以肺曲霉病和肺隐球菌病为多见,影像学表现主要以肺部肿块影和渗出性病变为主。肺真菌病影像学表现多种多样、缺乏特征性,诊断应尽早取得病理学依据。     

肝病患者真菌感染的特点及耐药性分析

目的分析肝病患者真菌感染的特点和耐药性,为临床预防真菌感染和合理用药提供依据。方法回顾分析2011年1月—2014年6月临床分离的1472株真菌的组成及耐药性。结果肝病患者真菌感染的主要标本来源是痰、中段尿和腹水。菌种以白假丝酵母菌为主(54.96%),其次是烟曲霉菌(13.25%),热带假丝酵母菌和光滑假丝酵母菌分别为10.39%和10.26%。丝状真菌对抗真菌药的耐药率明显高于酵母菌(P=0.000)。对氟康唑的耐药率较高的为烟曲霉菌(100%)、克柔假丝酵母菌(81.67%)和光滑假丝酵母菌(24.50%),克柔假丝酵母菌对伊曲康唑的耐药率达16.67%,白假丝酵母菌的药物敏感性较好。结论肝病患者真菌感染以白假丝酵母菌为主,其次是烟曲霉菌。不同种的真菌对不同抗真菌药的敏感性不同,临床应根据患者真菌感染和耐药特点合理治疗。     

Allergic bronchopulmonary fungal disease caused by Bipolaris and Curvularia

Abstract Allergic bronchopulmonary fungal disease (ABPFD) usually manifests in asthmatics as allergic bronchopulmonary aspergillosis. In a few instances other fungi have been implicated. Serological testing in Western Australia between 1979 and 1986 revealed precipitins to Bipolaris and Curvularia species in 40 of 503 patients tested. Eight of these were patients with ABPFD due to Bipolaris and/or Curvularia and are reported here. Geographical location appeared to be significant as seven of eight of those with ABPFD (and at least 18 of 40 with positive serology) were living in the more remote and sub-tropical northern part of the state. ABPFD due to fungi other than Aspergillus species may be more common than previously recognised and further epidemiological assessment is warranted. (Aust NZ J Med 1991; 21: 871–874.)     

Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens

The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity.In nature, plants are constantly exposed to a large number of pathogens including fungi, bacteria, and viruses. In response, plants have evolved multiple layers of defense mechanisms to resist the pathogen attack (1). The first line of plant defense against fungi is penetration resistance that is achieved by localized cell wall appositions (CWAs), also called papillae, on an inner surface of cell walls at the site of fungal penetration (2). CWAs consist primarily of callose (β-1,3-glucan), lignin, cell wall proteins, and reactive oxygen species (2–4). The focal deposition of these elements in papillae appears to be an early and essential factor in plant penetration resistance (5).Studies on the genetic basis of penetration resistance have revealed that entry control of A. thaliana against nonadapted powdery mildews largely depends on several PENETRATION (PEN) genes (PEN1, PEN2, and PEN3). All three PEN proteins are also recruited to attempted fungal penetration sites (6–11). Intriguing findings show that the focal accumulation of PEN1 and PEN3 occurs outside the plasma membrane and within papillae or haustorial encasements (3, 11, 12). Disruption of actin cytoskeleton by pharmacological inhibitors blocks PEN3–GFP accumulation at most penetration sites, but has a lesser effect on the recruitment of GFP–PEN1 to these sites (11), suggesting that transport pathways mediating PEN1 and PEN3 recruitment and export to the apoplastic papillae are distinct.Accumulation of dynamically moving cytoplasm near the pathogen penetration site is the most striking and microscopically visible early response in epidermal cells (2). The secretory vesicles and organelles, including peroxisomes, Golgi, mitochondria, and the nucleus, also move toward penetration sites (7, 13). In addition to the deposition of cell wall reinforcements and focal accumulation of penetration-related proteins such as PEN3, the accretion of cytoplasm and organelles at sites of attempted fungal penetration involves reorganization of actin cytoskeleton, which forms a radial array focused on penetration site (10, 11, 14–18). Consistent with this finding, disruption of AFs hampers penetration resistance, leading to increased penetration frequency by various fungal and oomycete pathogens (15–17, 19). However, the mechanisms that drive AF dynamics and active transport of cellular components toward sites of attempted pathogen penetration remain elusive. Myosins are molecular motors responsible for AF-based motility (20). Recently, plant class XI myosins were implicated in the organization of actin cytoskeleton, organelle and vesicle transport, cell expansion, and plant growth (21–27). Although none of the individual myosin gene knockouts produces plant growth defects (22), progressive elimination of two to four highly expressed myosins results in concomitant reduction in cell and plant size (23, 24). However, relatively little is known about the functions of myosins in plant–pathogen interactions (28, 29).Using pharmacological and genetic approaches to disrupt myosin function in Arabidopsis, we show that transient assembly and polarization of actin filament (AF) bundles toward the fungal penetration site are regulated by myosin motors. Furthermore, we demonstrate that plant myosins contribute to focal aggregation of a battery of cellular defense activities at the infection site and papillary deposition of cell wall appositions of lignin-like compounds, callose and PEN1, and are required for plant penetration resistance.     

多发性骨髓瘤合并侵袭性真菌感染的临床特点及易感因素分析

目的 了解多发性骨髓瘤(MM)患者合并侵袭性真菌感染(IFI)的临床特点及易感因素.方法 回顾357例在我院住院诊治的MM患者,记录是否合并 IFI、一般临床资料、并发病、抗真菌治疗以及疗效和毒副作用.结果 44例(12.3%)患者在治疗过程中曾发生IFI,其中3例(6.8%)为确诊病例,8例(18.1%)为临床诊断,33例(75.0%)为拟诊.44例患者中,10例(22.7%)处于诱导化疗时出现真菌感染;4例(9.1%)为平台期;27例(61.4%)处于疾病进展状态;3例(6.8%)在行自体造血干细胞移植的过程中发生真菌感染.感染部位以肺部最常见,占50.O%.两性霉素B、伏立康唑、伊曲康唑、卡泊芬净、氟康唑的有效率分别为83.3%、75.O%、78.9%、75.O%和57.1%.各种抗真菌药物之间疗效比较差异无统计学意义(P=0.493).根据多因素分析,合并糖尿病(P=0.035,OR 2.527,95%CI 1.005～6.052),接受透析治疗(P=0.022,OR 2.768,95%CI 1.161～6.600)、粒细胞缺乏持续时间超过1周(P:0.019,OR 3.215,95%CI 1.200～7.407),之前是否使用广谱抗生素治疗(P=0.009,OR 3.350,95%CI 1.353～8.295),是否使用氟达拉滨(P=0.001,OR 4.669,95%CI1.813-12.023)差异有统计学意义.结论 MM患者是侵袭性真菌感染的高危人群,肺部是其最常见的感染部位,4种抗真菌药的疗效相当,合并糖尿病、化疗同时接受透析治疗、长时间粒细胞缺乏、广谱抗生素的应用、以及含有氟达拉滨的治疗是MM合并IFI的易感因素.     

Chronic endogenous fungal endophthalmitis: diagnostic and treatment challenges: A case report

Rationale:Endogenous fungal endophthalmitis (EFE) is a sight-threatening complication of systemic fungemia. As the prevalence rises, treatment remains a challenge especially when there is a failure in first-line treatment or drug-resistant fungus. This case report studies a case of chronic EFE, focusing on the diagnostic procedures, treatment options, monitoring parameters and the treatment outcomePatient concerns:A 64-year-old man with underlying well controlled diabetes mellitus was treated with 2 weeks’ course of intravenous antifungal fluconazole for pyelonephritis as his blood culture grew Candida albicans. Concurrently, he complained of 3 months of bilateral painless progressive blurring of vision. At presentation, his visual acuity (VA) was light perception both eyes. Ocular examination revealed non granulomatous inflammation with dense vitritis of both eyes.Diagnosis:He was diagnosed with EFE but the condition responded poorly with the medications.Interventions:He was treated with intravitreal (IVT) amphotericin B and fluconazole was continued. Vitrectomy was performed and intraoperative findings included bilateral fungal balls in the vitreous and retina with foveal traction in the left eye. Postoperatively, vision acuity was 6/24, N8 right eye and 2/60, N unable for left eye with extensive left macular scar and hole. Vitreous cultures were negative. He received multiple IVT amphotericin B and was started on topical steroid eye drops for persistent panuveitis with systemic fluconazole. Ocular improvement was seen after switching to IVT and topical voriconazole. Despite this, his ocular condition deteriorated and he developed neovascular glaucoma requiring 3 topical antiglaucoma agents. Panretinal photocoagulation was subsequently performed.Outcomes:At 3 months’ follow-up, his vision acuity remained at 6/24 for right eye and 2/60 for the left eye. There was no recurrence of inflammation or infection in both eyes.Lessons:Voriconazole could serve as a promising broad spectrum tri-azole agent in cases of failure in first-line treatment or drug-resistant fungus.     

Impact of invasive fungal disease on the chemotherapy schedule and event-free survival in acute leukemia patients who survived fungal disease: a case-control study

Patients with acute leukemia who initially survive invasive fungal disease must receive chemotherapy or go on to transplant. Many centers change subsequent chemotherapy to decrease the risk of fungal reactivation. This case-control study compared acute leukemia patients (n=28) who developed a proven or probable fungal disease and survived four weeks later, to patients who did not (n=78), and assessed the impact of fungal disease on the chemotherapy regimens, and overall and event-free survival. Chemotherapy changes (i.e. delays, dose-reduction) were more frequent in the fungal (68%) than in the control group (24%) (P<0.001). Although there was no difference in overall and event-free survival between groups, they were both lower for proven fungal disease cases when compared to controls (HR 2.4, 95% CI 1.1-1.5, and HR 2.9, 95% CI 1.4-5.6, respectively). Patients with invasive fungal disease, even though they initially survive, undergo significant changes to their chemotherapy therapy. This impacts on the survival of patients with proven fungal disease.