Peptide amphipathy: a new strategy in design of potential insecticides

A 30-residue peptide [YAA(KALA)₆LAA] with an amphipathic helix repeat unit of Lys-Ala-Leu-Ala (KALA) was synthesized as both the l - and the d -isomer. The peptide was shown to form α-helices and lyse lipid vesicles in a pH dependent fashion. The calculated helical amphipathic moment is + 1.19 kcal/ residue and the mean residue hydrophobicity is +0.4 kcal/residue. The formation of α-helices as the pH is increased is similar to poly-lysine, yielding a pK of 10.2. Though not toxic when fed to insects, KALA killed Spodoptera frugiperda cells at low doses and Manduca sexta larvae when injected.     

Urate Nephropathy from Tumor Lysis Syndrome in an Undiagnosed Case of Prostate Cancer

Prostate cancer can masquerade as just normocytic anemia and thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), or tumor lysis syndrome (TLS). We are reporting an intriguing case of metastatic prostate cancer which remained undiagnosed until the patient showed signs of tumor lysis syndrome (TLS), leading to urate nephropathy requiring urgent hemodialysis. Tumor lysis syndrome is an oncological emergency but an exceedingly rare complication in non-hematological malignancies, including prostate cancer. It is challenging to recognize features of TLS in a case such as this with an unknown diagnosis. In the case of an established diagnosis of malignancy, however, checking baseline renal function, uric acid, lactate dehydrogenase (LDH), potassium, and phosphate to monitor for TLS as well as considering urate lowering therapy can help prevent adverse outcomes.     

侵袭性NK细胞白血病发生肿瘤溶解综合征1例报告及文献复习

目的:提高对侵袭性NK细胞白血病和肿瘤溶解综合征(Tumour Lysis Syndrome,TLS)的认识。方法:报告1例侵袭性NK细胞白血病患者在治疗过程中发生TLS,介绍目前关于TLS的研究进展。结果:确诊侵袭性NK细胞白血病1例,予CHOP方案治疗。在治疗过程中患者出现高钾、高磷、高尿酸及尿素氮血症及肌酐升高等肿瘤溶解综合征的特点,经积极对症、支持治疗得以纠正。血象恢复后复查骨髓达完全缓解。结论:侵袭性NK细胞白血病是一种少见疾病,预后不良;而治疗中发生肿瘤溶解综合征,使结局进一步恶化.但若能早期预测、发现TLS,并予积极的治疗,能得到好的转归。     

Molecular mechanisms and functions of pyroptosis,inflammatory caspases and inflammasomes in infectious diseases

Cell death is a fundamental biological phenomenon that is essential for the survival and development of an organism. Emerging evidence also indicates that cell death contributes to immune defense against infectious diseases. Pyroptosis is a form of inflammatory programmed cell death pathway activated by human and mouse caspase-1, human caspase-4 and caspase-5, or mouse caspase-11. These inflammatory caspases are used by the host to control bacterial, viral, fungal, or protozoan pathogens. Pyroptosis requires cleavage and activation of the pore-forming effector protein gasdermin D by inflammatory caspases. Physical rupture of the cell causes release of the pro-inflammatory cytokines IL-1β and IL-18, alarmins and endogenous danger-associated molecular patterns, signifying the inflammatory potential of pyroptosis. Here, we describe the central role of inflammatory caspases and pyroptosis in mediating immunity to infection and clearance of pathogens.     

Complete Lysis of Left Ventricular Giant Thrombus with Fibrinolytic Therapy in Clopidogrel Resistant Patient

Sixty-year-old woman admitted with dyspnea and cough. Three weeks ago she underwent primary stenting for acute anterior myocardial infarction and recieved antiplatelet therapy (clopidogrel). Echocardiography and left ventriculography revealed left ventricular segmental dysfunction at anterolateral-apical region but no thrombus. On last admission, despite the clopidogrel therapy, echocardiography showed giant-partly mobil thrombus obliterated half of the left ventricle. Slow infusion of thrombolytic therapy was given and complete lysis occurred with uneventful course. Disclosure of such a rapidly evolving giant left ventricular thrombus in the clopidogrel non-responder is a rare clinical problem with potentially catastrophic consequences. Slow infusion of thrombolytic therapy may be effective and life saving.     

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon—Shigella IpaC, Salmonella SipC, or Chromobacterium CipC—determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established.Intracellular bacterial pathogens use a variety of elaborate means to survive within host cells. Postinvasion, some such as Legionella, Salmonella, and Chlamydia species modify bacteria-containing vacuoles to avoid death via phagosomal acidification or lysosomal fusion. Others, including Shigella, Listeria, Rickettsia, and Burkholderia species, rapidly escape from phagosomes into the cytosol of infected cells. Although escape from phagosomes by the classic intracytoplasmic Gram-positive bacterium Listeria monocytogenes is well understood (1), much less is known regarding how Gram-negative pathogens, including the model professional intracytoplasmic Shigella species, enter the cytosol.During the course of an infection, many Gram-negative pathogens, including Shigella, Salmonella, enteropathogenic Escherichia coli, and Yersinia species, use type III secretion systems (T3SSs) as injection devices to deliver multiple virulence proteins, referred to as effectors, directly into the cytosol of infected cells (2). T3SSs are composed of ∼20 proteins and sense host cell contact via a tip complex at the distal end of a needle filament, which then acts as a scaffold for the formation of a translocon pore in the host cell membrane. Although components of their type III secretion apparatus (T3SA) are relatively well conserved, each pathogen delivers a unique repertoire of effectors into host cells, likely accounting for the establishment of a variety of replicative niches. For example, Salmonella and Shigella secreted effectors promote the uptake of these bacteria into nonphagocytic cells, whereas those from Yersinia inhibit phagocytosis by macrophages.All four pathogenic Shigella species—Shigella flexneri, Shigella sonnei, Shigella boydii, and Shigella dysenteriae—deliver ∼30 effectors into host cells, the majority of which are encoded on a large virulence plasmid (VP) alongside the genes for all of the proteins needed to form a T3SA (3). These secreted proteins play major roles in Shigella pathogenesis, including host cell invasion and modulation of innate immune response. One effector, IpgD, promotes the efficiency of Shigella phagosomal escape, although it is not absolutely required for this process (4). Interestingly, IpaB and IpaC, components of the Shigella translocon, the portion of the T3SA that inserts into the host cell membrane, have been implicated to mediate phagosomal escape based on the behavior of recombinant proteins (5–7). The physiologic relevance of these findings has not yet been directly addressed, as strains that lack either of these two proteins are completely impaired in the delivery of Shigella effectors into host cells (8).Here, using a reductionist approach, we directly tested a role for the Shigella translocon apparatus in phagosomal escape. Using an innovative reengineering approach, we introduced a functional effectorless Shigella T3SA into a nonpathogenic laboratory strain of DH10B E. coli. Remarkably, upon entry into host epithelial cells, these bacteria efficiently escape from phagosomes. This demonstrates for the first time, to our knowledge, in the context of an infection, a direct role for the Shigella T3SA in mediating vacuole lysis. Despite structural conservation across T3SS families, we further observed that, in the absence of any type III effectors, the Ysc T3SA mediates little to no Yersinia phagosomal escape, suggesting that not all injectisomes have equivalent functions. Lastly, by exploring the functional interchangeability of translocon components of the Shigella, Salmonella, and Chromobacterium T3SA, we demonstrate that one translocon protein controls the extent to which these intracellular pathogens escape into the cytosol of infected cells, thus demonstrating a major role for the T3SA in determining the site of the replicative niche of intracellular bacteria.     

双腔管冲洗引流并尿激酶溶解术治疗高血压脑出血

目的:探讨双腔管冲洗引流并尿激酶溶解术治疗高血压脑出血的方法及效果.方法:回顾性分析38例在CT定位钻孔抽吸部分血肿后行双腔管冲洗引流并尿激酶注入治疗的高血压脑出血病人的临床特点及疗效.结果: 血肿清除率高,病人恢复良好,死亡率低,并发症少.结论:双腔管冲洗引流并尿激酶溶解术是高血压脑出血的一种简单、有效的治疗方法.     

Lysis of pathogenic and nonpathogenic Entamoeba histolytica by human complement: methodological analysis

The effect of nonimmune human serum on Entamoeba histolytica trophozoites was studied: (a) using whole serum in the presence of Ca and Mg ions allowing complement activation via both the alternative and classical pathways or in the presence of MgEGTA permitting alternative pathway activation only; (b) using different E. histolytica isolates; (c) varying serum and trophozoite concentrations and the time of incubation; and (d) using three different methods to quantify lysis, i.e., microscopic inspection, flow cytometry and 111In release. All three methods yielded similar results, with flow cytometry being most sensitive in identifying membrane damage and 111In release being most valid in determining cell death. Microscopic analysis was reliable only when a chamber was used to calculate the number of complement treated cells in relation to the initial cell count. E. histolytica isolates were classified into three groups according to their susceptibility to lysis by complement: (i) pathogenic isolates after long term cultivation in vitro were susceptible; (ii) pathogenic isolates after recent in vivo passage were less susceptible; and (iii) nonpathogenic isolates were nearly unaffected by exposure to the alternative pathway alone. The extent of lysis of the various isolates correlated with the degree of complement consumption in the serum samples, suggesting that unlysed isolates did not activate complement under the conditions employed. In general, lysis of susceptible trophozoites increased with the serum concentration and with the time of incubation. However, when the trophozoite concentration was 10(6)/ml or higher, lysis no longer reflected complement susceptibility because of exhaustion of the complement supply.     

Eimeria tenella: quantitative in vitro and in vivo studies on the effects of mouse polyclonal and monoclonal antibodies on sporozoites

Murine, polyclonal and monoclonal antibodies, raised against sporozoites of Eimeria tenella, were tested for their ability to neutralize sporozoite infectivity in vitro and in vivo. Neutralization was effected via three mechanisms. Firstly, sporozoites fixed complement, at low titres, and lysis occurred by the alternative pathway of complement activation. Secondly, in the absence of complement activity, the murine heat-inactivated, hyperimmune antiserum neutralized sporozoites at relatively low titres. At high titres, even though sporozoites were agglutinated, neither the heat-inactivated hyperimmune antiserum nor the monoclonal antibody neutralized sporozoites. Finally, in the presence of complement and specific antibodies, at titres which by themselves would not neutralize sporozoites, neutralization was effected due to lysis via the classical pathway of complement activation.     

Exopolysaccharide defects cause hyper-thymineless death in Escherichia coli via massive loss of chromosomal DNA and cell lysis

Thymineless death in Escherichia coli thyA mutants growing in the absence of thymidine (dT) is preceded by a substantial resistance phase, during which the culture titer remains static, as if the chromosome has to accumulate damage before ultimately failing. Significant chromosomal replication and fragmentation during the resistance phase could provide appropriate sources of this damage. Alternatively, the initial chromosomal replication in thymine (T)-starved cells could reflect a considerable endogenous dT source, making the resistance phase a delay of acute starvation, rather than an integral part of thymineless death. Here we identify such a low-molecular-weight (LMW)-dT source as mostly dTDP-glucose and its derivatives, used to synthesize enterobacterial common antigen (ECA). The thyA mutant, in which dTDP-glucose production is blocked by the rfbA rffH mutations, lacks a LMW-dT pool, the initial DNA synthesis during T-starvation and the resistance phase. Remarkably, the thyA mutant that makes dTDP-glucose and initiates ECA synthesis normally yet cannot complete it due to the rffC defect, maintains a regular LMW-dT pool, but cannot recover dTTP from it, and thus suffers T-hyperstarvation, dying precipitously, completely losing chromosomal DNA and eventually lysing, even without chromosomal replication. At the same time, its ECA⁺ thyA parent does not lyse during T-starvation, while both the dramatic killing and chromosomal DNA loss in the ECA-deficient thyA mutants precede cell lysis. We conclude that: 1) the significant pool of dTDP-hexoses delays acute T-starvation; 2) T-starvation destabilizes even nonreplicating chromosomes, while T-hyperstarvation destroys them; and 3) beyond the chromosome, T-hyperstarvation also destabilizes the cell envelope.

Acute starvation for thymidine triphosphate (dTTP), one of the four precursors for DNA synthesis, is lethal in both bacterial and eukaryotic cells (1). Following a short resistance phase, the rapid death of thyA auxotrophs in media lacking thymine or thymidine (“T-starvation”) known as thymineless death (TLD) was first described in Escherichia coli (2, 3) and since then was extensively studied to identify the cause of lethality (1, 4, 5). Because the bulk of thymidine (dT) in any cell is used for chromosomal DNA synthesis, lack of dT was always assumed to cause some form of chromosomal damage, and hence the role of DNA repair pathways during T-starvation was the focus of intense investigation (6–9). These studies revealed that certain pathways, like double-strand break repair initiated by the RecBCD helicase/nuclease, Holliday junction resolution by RuvABC, and antirecombination activity of the UvrD helicase, keep cells alive during the resistance phase of T-starvation. Other events, like attempted single-strand gap repair initiated by the RecFOR complex, the function of the RecQ helicase and RecJ exonuclease, and SOS induction of the cell division inhibitor SulA, are detrimental for T-starved E. coli cells (8, 10–12). However, the thyA mutants of E. coli inactivated for all of the latter “toxic DNA repair pathways” still die by two orders of magnitude during T-starvation (8), indicating some other yet-to-be-identified major lethality factors.Since actively growing cells continuously require a lot of dT to replicate chromosomal DNA, existing replication forks were inferred to be the points of TLD pathology (7, 8, 13–15). Indeed, T-starvation severely inhibits chromosomal DNA replication (15) and is associated with accumulation of single-stranded DNA, suggesting generation of single-strand (ss) gaps by attempted replication in the absence of dT (7, 16). These ss-gaps induce the SOS response (7, 8, 17), which contributes to the pathology of TLD by induction of the SulA cell division inhibitor (8). Also, replication initiation spike in the T-starved cells triggers the destruction of the origin-centered chromosomal subdomain during TLD, suggesting that it is the demise of the nascent replication bubbles, rather than the existing replication forks, that eventually kills the chromosome (15, 17).Although the thyA mutants cannot synthesize dT, they grow normally if supplemented with exogenous dT/T. Upon removal of dT from the growth medium, the E. coli thyA strain has a two-generation-long resistance phase (also called the lag phase) (1), when the colony-forming unit (CFU) titer of the culture stays constant (Fig. 1 A, Top). This is followed by the rapid exponential death (RED) phase, when the CFU titer falls by approximately three orders of magnitude within several hours (Fig. 1 A, Top).Open in a separate windowFig. 1.A significant endogenous pool of LMW dT decreases during T-starvation. (A) The phenomenon of TLD in E. coli suggests accumulation of chromosomal damage during the resistance phase (green) that would later kill cells during the RED phase (red). The data are adapted from ref. 16. Henceforth, the data are means (n ≥ 3) ± SEM. Cultures were grown at 37 °C in the presence of dT, which was removed at time = 0, while incubation in the growth medium continued. Top, cell death begins after 1-h-long resistance phase, during which the culture titer is stable. Bottom, during the same first hour without dT, cells manage to synthesize the amount of DNA equal to half of what they already had before dT removal. However, during the RED phase genomic DNA is gradually lost. (B) Scheme of 50% methanol fractionation of the intracellular thymidine into HMW dT (the dT content of the chromosomal DNA) and LMW dT. (C) A 0.7% agarose gel analysis of the HMW and LMW fractions of the 50% methanol-treated cells, as well as pure LB treated the same way, for DNA and RNA content (staining with ethidium bromide). Inverse images of stained gels are shown, in which the indicated samples were either incubated in the buffer or with the indicated enzyme (DNase I for the top gel, RNase A for the bottom gel). (D) The size of the LMW-dT pool, normalized to the HMW-dT content of the chromosome, either during normal growth in dT-supplemented medium or during T-starvation. Thymidine was removed at time = 0. The strain is KKW58.An obvious explanation for the resistance phase is existence of an intracellular source of dT to support slow replication; however, chromosomal DNA amount was consistently reported to remain flat during TLD (15, 18–20). Besides, the recent systematic test of potential candidates for a source of dT or its analogs supporting the resistance phase returned empty-handed (16). Thus, the mechanisms behind the initial resistance to T-starvation, followed by the sudden shift to the RED phase remain unclear, leading to a reasonable assumption that the resistance phase is an integral part of the TLD phenomenon, during which chromosomal damage accumulates until it becomes irreparable, ushering the RED phase (1, 5). Specific early events during the resistance phase of TLD that would later turn poisonous during the RED phase were proposed to be futile incorporation–excision cycles (1, 21), ss-gap accumulation causing the SOS induction (1, 8, 16), futile fork breakage-repair cycles (16, 22), and overinitiation from the origin (5, 15).Two recent observations, in combination with an old popular TLD explanation, further support the idea of the resistance phase as the TLD period during which chromosomal damage accumulates without affecting viability for the time being. First, the resistance phase coincides with accumulation of double-strand breaks in the chromosome, which then paradoxically disappear during the RED phase (7, 16). Second (and in contrast to the reports mentioned above of constant chromosomal DNA amount during T-starvation) (15, 18–20), we have found that during the resistance phase the amount of the chromosomal DNA actually increases ∼1.5 times over the prestarvation level, but then the chromosomal DNA is apparently destabilized during the RED phase, since it is slowly reduced to the original level (16) (Fig. 1 A, Bottom). Therefore, both the apparent chromosomal replication and the significant chromosome fragmentation during the resistance phase could lead to accumulation of chromosomal damage (SOS induction is an indicator of this accumulation) (7).On the other hand, the early DNA synthesis and the resistance phase in T-starved cells could reflect the existence of a source of dT, available early on during T-starvation, that fuels the initial DNA accumulation and delays viability loss until this pool is exhausted. In other words, the resistance phase could simply postpone TLD, rather than being an integral part of it. Previously, we have tested the two obvious high-molecular-weight (HMW) dT sources, namely, the stable RNAs and the chromosomal DNA, but found that incapacitation of neither one reduced the resistance phase or precluded the early DNA synthesis during T-starvation (16). Thus, the question of whether the resistance phase is a part of the TLD phenomenon remains unresolved.In the current study, we investigated a seemingly remote possibility of a substantial low-molecular-weight (LMW)-dT pool supporting the resistance phase of T-starvation in E. coli. While the bulk of dTTP in E. coli immediately incorporates into the chromosomal DNA, a fraction of dTTP is recruited into the dTDP-hexose pool (23), to participate in the synthesis of the exopolysaccharide (EPS) capsule, made of core lipopolysaccharide (LPS) (24), O-antigen (OA) (25), and enterobacterial common antigen (ECA) in E. coli (26). The first step of this recruitment is to conjugate dTTP with glucose; the hexose moiety of the resulting dTDP-glucose then undergoes several modifications, before eventually incorporating into oligosaccharide precursors of the outer antigens, while the activating dTDP handle is released back into the DNA precursor pools (26). We ignored LMW dT before because, if the total dT content of the chromosomal DNA is taken for 100%, the pool of dTTP constitutes ∼0.7% of it, while dTDP-glucose (unresolved from other dTDP-hexoses?) adds only another 2.4% (27). No more LMW-dT species are known in the cell, so the total expected LMW-dT pool comes to ∼3% of the total dT content of the chromosomal DNA, not nearly enough to support the resistance phase with its ∼50% increase in the chromosomal DNA mass (Fig. 1A).To investigate the role of the LMW-dT pool in TLD, we started by developing a simple protocol to extract the LMW-dT pool from growing cells and to compare it to the (HMW) chromosomal dT content. Here we show that early on during T-starvation the pool of dTDP-sugars becomes the major source of dTTP for the chromosomal DNA replication. This unexpected rebalancing of the dTTP pool with the help of cell envelope metabolism delays TLD and prevents T-hyperstarvation, a significantly more lethal phenomenon accompanied by complete chromosome destruction and cell lysis.