Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca2+-Mediated Activation of Esterase Activity.

The presence of Ca2+ ions is known to facilitate the activity of trypsin-like serine proteases via structural stabilization against thermal denaturation and autolysis. Herein, we report a new and hidden regulatory role of Ca2+ in the catalytic pathways of trypsin and α-chymotrypsin under physiological conditions. We discovered that macromolecular crowding promotes spontaneous homotypic condensation of trypsin via liquid-liquid phase separation to yield membraneless condensates over a broad range of concentrations, pH, and temperature, which are stabilized by multivalent hydrophobic interactions. Interestingly, we found that Ca2+ binding in the calcium binding loop reversibly regulates the condensation of trypsin and α-chymotrypsin. Spontaneous condensation effectively prevents autolysis of trypsin and preserves its native-like esterase activity for a prolonged period of time. It has also been found that phase-separated trypsin responds to Ca2+-dependent activation of its esterase activity even after 14 days of storage while free trypsin failed to do so. The present study highlights an important physiological aspect by which cells can spatiotemporally regulate the biocatalytic efficacy of trypsin-like serine proteases via Ca2+-signaling.

Effect of humidity on postmortem changes in rats.

IMPORTANCE: In veterinary forensic science, accurately determining the postmortem interval (PMI) is crucial for identifying the causes of animal deaths. Autolysis, a significant postmortem process, influences PMI estimation, but its relationship with humidity is not well understood. OBJECTIVE: This study aimed to improve the accuracy of PMI estimates in veterinary forensic cases by looking into how different humidity levels affect autolysis in different organs of rats. METHODS: The study involved 38 male rats, examining histopathological changes in their heart, liver, and pancreas. These organs were subjected to controlled humidity levels (20%, 55%, and 80%) at a constant 22°C. Tissue samples were collected at several intervals (0 h, 12 h, 24 h, 3 days, and 8 days) for comprehensive analysis. RESULTS: Distinct autolytic characteristics in animal organs emerged under varying humidity conditions. The low-humidity environment rapidly activated autolysis more than the high-humidity environment. In addition, it was found that lower humidity caused nuclear pyknosis, cytoplasmic disintegration, and myofiber interruption. The liver, in particular, showed portal triad aggregation and hepatocyte individuation. The pancreas experienced cell fragmentation and an enlarged intracellular space. High humidity also caused the loss of striations in cardiac tissues, and the liver showed vacuolation. Under these conditions, the pancreas changed eosinophilic secretory granules. CONCLUSIONS AND RELEVANCE: The study successfully established a clear connection between the autolytic process in PMIs and relative humidity. These findings are significant for developing a more accurate and predictable method for PMI estimation in the field of veterinary forensic science.

An induced mutation of ABC-transporter component VraF(K84E) contributes to vancomycin resistance and virulence in Staphylococcus aureus strain MW2.

Staphylococcus aureus is a notorious pathogen responsible for various severe diseases. Due to the emergence of drug-resistant strains, the prevention and treatment of S. aureus infections have become increasingly challenging. Vancomycin is considered to be one of the last-resort drugs for treating most methicillin-resistant S. aureus (MRSA), so it is of great significance to further reveal the mechanism of vancomycin resistance. VraFG is one of the few important ABC (ATP-binding cassette) transporters in S. aureus that can form TCS (two-component systems)/ABC transporter modules. ABC transporters can couple the energy released from ATP hydrolysis to translocate solutes across the cell membrane. In this study, we obtained a strain with decreased vancomycin susceptibility after serial passaging and selection. Subsequently, whole-genome sequencing was performed on this laboratory-derived strain MWA2 and a novel single point mutation was discovered in vraF gene, leading to decreased sensitivity to vancomycin and daptomycin. Furthermore, the mutation reduces autolysis of S. aureus and downregulates the expression of lytM, isaA, and atlA. Additionally, we observed that the mutant has a less net negative surface charge than wild-type strain. We also noted an increase in the expression of the dlt operon and mprF gene, which are associated with cell surface charge and serve to hinder the binding of cationic peptides by promoting electrostatic repulsion. Moreover, this mutation has been shown to enhance hemolytic activity, expand subcutaneous abscesses, reflecting an increased virulence. This study confirms the impact of a point mutation of VraF on S. aureus antibiotic resistance and virulence, contributing to a broader understanding of ABC transporter function and providing new targets for treating S. aureus infections.

Perfusion Pressure and the Histology of Brain Death: A Unique Case in an Infant Maintained on Life Support.

Brain death is a not uncommon phenomena in the adult and pediatric population. Most cases are removed from life support soon after brain death is declared. Less commonly, systemic perfusion is maintained by life support for some time after neurologic function stops. These cases present uncommon opportunities to explore the histology of necrosis and autolysis in the context of global hypoxic ischemic damage. Here, we describe the unusual case of an infant maintained on life support for 2 weeks after brain death was declared with an emphasis on the resulting gross and histologic findings including a discussion of their underlying physiology.

[Manipulation of isocitrate dehydrogenase genes affects the anti-autolytic ability of lager yeast].

Yeast autolysis affects the flavor and quality of beer. The regulation of yeast autolysis is a need for industrial beer production. Previous studies on brewer's yeast autolysis showed that the citric acid cycle-related genes had a great influence on yeast autolysis. To explore the contribution of isocitrate dehydrogenase genes in autolysis, the IDP1 and IDP2 genes were destroyed or overexpressed in typical lager yeast Pilsner. The destruction of IDP1 gene improved the anti-autolytic ability of yeast, and the anti-autolytic index after 96 h autolysis was 8.40, 1.5 times higher than that of the original strain. The destruction of IDP1 gene increased the supply of nicotinamide adenine dinucleotide phosphate (NADPH) and the NADPH/NADP+ ratio was 1.94. After fermentation, intracellular ATP level was 1.8 times higher than that of the original strain, while reactive oxygen species (ROS) was reduced by 10%. The destruction of IDP2 gene resulted in rapid autolysis and a decrease in the supply of NADPH. Anti-autolytic index after 96 h autolysis was 4.03 and the NADPH/NADP+ ratio was 0.89. After fermentation, intracellular ATP level was reduced by 8% compared with original strain, ROS was 1.3 times higher than that of the original strain. The results may help understand the regulation mechanism of citric acid cycle-related genes on yeast autolysis and provide a basis for the selection of excellent yeast with controllable anti-autolytic performance.

DipM controls multiple autolysins and mediates a regulatory feedback loop promoting cell constriction in Caulobacter crescentus.

Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their representative DipM, a factor promoting cell division in Caulobacter crescentus. We show that the LytM domain of DipM interacts with multiple autolysins, including the soluble lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. Its crystal structure reveals a conserved groove, which is predicted to represent the docking site for autolysins by modeling studies. Mutations in this groove indeed abolish the function of DipM in vivo and its interaction with AmiC and SdpA in vitro. Notably, DipM and its targets SdpA and SdpB stimulate each other's recruitment to midcell, establishing a self-reinforcing cycle that gradually increases autolytic activity as cytokinesis progresses. DipM thus coordinates different peptidoglycan-remodeling pathways to ensure proper cell constriction and daughter cell separation.

Alkyl-quinolone-dependent quorum sensing controls prophage-mediated autolysis in Pseudomonas aeruginosa colony biofilms.

Pseudomonas aeruginosa is a model quorum sensing (QS) pathogen with three interconnected QS circuits that control the production of virulence factors and antibiotic tolerant biofilms. The pqs QS system of P. aeruginosa is responsible for the biosynthesis of diverse 2-alkyl-4-quinolones (AQs), of which 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) function as QS signal molecules. Transcriptomic analyses revealed that HHQ and PQS influenced the expression of multiple genes via PqsR-dependent and -independent pathways whereas 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) had no effect on P. aeruginosa transcriptome. HQNO is a cytochrome bc 1 inhibitor that causes P. aeruginosa programmed cell death and autolysis. However, P. aeruginosa pqsL mutants unable to synthesize HQNO undergo autolysis when grown as colony biofilms. The mechanism by which such autolysis occurs is not understood. Through the generation and phenotypic characterization of multiple P. aeruginosa PAO1 mutants producing altered levels of AQs in different combinations, we demonstrate that mutation of pqsL results in the accumulation of HHQ which in turn leads to Pf4 prophage activation and consequently autolysis. Notably, the effect of HHQ on Pf4 activation is not mediated via its cognate receptor PqsR. These data indicate that the synthesis of HQNO in PAO1 limits HHQ-induced autolysis mediated by Pf4 in colony biofilms. A similar phenomenon is shown to occur in P. aeruginosa cystic fibrosis (CF) isolates, in which the autolytic phenotype can be abrogated by ectopic expression of pqsL.

Manipulation of isocitrate dehydrogenase genes affects the anti-autolytic ability of lager yeast / 生物工程学报

Yeast autolysis affects the flavor and quality of beer. The regulation of yeast autolysis is a need for industrial beer production. Previous studies on brewer's yeast autolysis showed that the citric acid cycle-related genes had a great influence on yeast autolysis. To explore the contribution of isocitrate dehydrogenase genes in autolysis, the IDP1 and IDP2 genes were destroyed or overexpressed in typical lager yeast Pilsner. The destruction of IDP1 gene improved the anti-autolytic ability of yeast, and the anti-autolytic index after 96 h autolysis was 8.40, 1.5 times higher than that of the original strain. The destruction of IDP1 gene increased the supply of nicotinamide adenine dinucleotide phosphate (NADPH) and the NADPH/NADP+ ratio was 1.94. After fermentation, intracellular ATP level was 1.8 times higher than that of the original strain, while reactive oxygen species (ROS) was reduced by 10%. The destruction of IDP2 gene resulted in rapid autolysis and a decrease in the supply of NADPH. Anti-autolytic index after 96 h autolysis was 4.03 and the NADPH/NADP+ ratio was 0.89. After fermentation, intracellular ATP level was reduced by 8% compared with original strain, ROS was 1.3 times higher than that of the original strain. The results may help understand the regulation mechanism of citric acid cycle-related genes on yeast autolysis and provide a basis for the selection of excellent yeast with controllable anti-autolytic performance.

Transcriptomic analysis reveals process of autolysis of Kluyveromyces marxianus in vacuum negative pressure and the higher temperature

Kluyveromyces marxianus is expected to be used in the production of yeast extracts due to its good fermentation ability and nutritional properties. Yeast autolysis is a key process to produce yeast extract and vacuum negative pressure stress can be used as an effective way to assist autolysis. However, the molecular mechanism of initiating Kluyveromyces marxianus autolysis induced by vacuum negative pressure and the higher temperature is still unclear. In this study, RNA-seq technology was performed mainly to analyze autolytic processes in Kluyveromyces marxianus strains. Considerable differentially expressed genes (DEGs) of downregulation were significantly enriched in 7 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to synthesis and transport of RNA and ribosome, which indicated that abnormal protein translations had already occurred in autolytic process. Interestingly, due to obvious change of related DEGs, endoplasmic reticulum-associated degradation (ERAD) and autophagy were activated and cell wall integrity pathway was hindered. Under the continuous influence of the external stress environment, the long-term changes of the above pathways triggered a vicious circle of gradual damage to yeast cells, which is the main cause of yeast autolysis. These results may provide important clues for the in-depth interpretation of the yeast autolytic mechanism.(AU)

Sublethal HPH treatment is a sustainable tool that induces autolytic-like processes in the early gene expression of Saccharomyces cerevisiae.

Sublethal HPH treatments have been demonstrated to impact the technological properties and functions of treated microorganisms by inducing specific enzymes/genes or modulating membrane structures and inducing autolysis. In this work, the early effects of a 100 MPa HPH treatment on the winery starter Saccharomyces cerevisiae ALEAFERM AROM grown in synthetic must were assessed. While there were no differences in cell cultivability during the first 48 h between treated and untreated cells, a reduction in volatile metabolites released by HPH-treated cells during the first 2 h was observed. This reduction was only temporary since after 48 h, volatile molecules reached similar or even higher concentrations compared with the control. Moreover, the gene expression response of HPH-treated cells was evaluated after 1 h of incubation and compared with that of untreated cells. A massive rearrangement of gene expression was observed with the identification of 1220 differentially expressed genes (DEGs). Most of the genes related to energetic metabolic pathways and ribosome structure were downregulated, while genes involved in ribosome maturation, transcription, DNA repair, response to stimuli and stress were upregulated. These findings suggest that HPH induces or promotes an autolytic-like behaviour that can be exploited in winemaking.

Ryanodine receptors mediate high intracellular Ca2+ and some myocyte damage following eccentric contractions in rat fast-twitch skeletal muscle.

Eccentric contractions (ECC) facilitate cytosolic calcium ion (Ca2+) release from the sarcoplasmic reticulum (SR) and Ca2+ influx from the extracellular space. Ca2+ is a vital signaling messenger that regulates multiple cellular processes via its spatial and temporal concentration ([Ca2+]i) dynamics. We hypothesized that 1) a specific pattern of spatial/temporal intramyocyte Ca2+ dynamics portends muscle damage following ECC and 2) these dynamics would be regulated by the ryanodine receptor (RyR). [Ca2+]i in the tibialis anterior muscles of anesthetized adult Wistar rats was measured by ratiometric (i.e., ratio, R, 340/380 nm excitation) in vivo bioimaging with Fura-2 pre-ECC and at 5 and 24 h post-ECC (5 × 40 contractions). Separate groups of rats received RyR inhibitor dantrolene (DAN; 10 mg/kg ip) immediately post-ECC (+DAN). Muscle damage was evaluated by histological analysis on hematoxylin-eosin stained muscle sections. Compared with control (CONT, no ECC), [Ca2+]i distribution was heterogeneous with increased percent total area of high [Ca2+]i sites (operationally defined as R ≥ 1.39, i.e., ≥1 SD of mean control) 5 h post-ECC (CONT, 14.0 ± 8.0; ECC5h: 52.0 ± 7.4%, P < 0.01). DAN substantially reduced the high [Ca2+]i area 5 h post-ECC (ECC5h + DAN: 6.4 ± 3.1%, P < 0.01) and myocyte damage (ECC24h, 63.2 ± 1.0%; ECC24h + DAN: 29.1 ± 2.2%, P < 0.01). Temporal and spatially amplified [Ca2+]i fluctuations occurred regardless of DAN (ECC vs. ECC + DAN, P > 0.05). These results suggest that the RyR-mediated local high [Ca2+]i itself is related to the magnitude of muscle damage, whereas the [Ca2+]i fluctuation is an RyR-independent phenomenon.

Inhibition of ultraviolet-induced sea cucumber (Stichopus japonicus) autolysis by maintaining coelomocyte intracellular calcium homeostasis.

Apoptosis plays a critical role in sea cucumber autolysis. To investigate the ultraviolet (UV)-induced apoptosis, sea cucumbers with and without injection of BAPTA-AM (cytosolic calcium chelator) were exposed to UV (15 W/m2) for 30 min. The results showed that UV irradiation caused several changes in sea cucumber coelomocytes, including calcium imbalance, abnormal morphology of endoplasmic reticulum, upregulation of pro-apoptotic proteins CRT, CHOP, and caspases 9 and 3, and downregulation of anti-apoptotic protein Bcl-2. A comparison between the two groups showed that injection of the calcium chelator into sea cucumbers helped maintain coelomocyte intracellular calcium homeostasis and suppressed other abnormal changes caused by ER stress, indicating apoptosis in sea cucumbers is mediated by calcium imbalance and follows the activation of the ER stress pathway. Therefore, this study broadens understanding of the apoptotic mechanism involved in sea cucumber autolysis, which is helpful in developing preservative agents for sea cucumbers.

The exogenous autophagy inducement alleviated the sea cucumber (Stichopus japonicus) autolysis with exposure to stress stimuli of ultraviolet light.

BACKGROUND: Autolysis is the most important restrictive factor for the live sea cucumber trade and commercial transportation. Thus, it is essential to investigate the mechanism of autolysis activation or deactivation in the sea cucumber. In this study, monodansylcadaverine staining and Western blotting experiment methods indicated the implication of autophagy in the ultraviolet (UV) exposed sea cucumbers. The health condition was observed after the sea cucumbers (Stichopus japonicus) were gastric perfusion with autophagic inhibitor (3-methyladenine) or inducer (rapamycin) and exposure to UV light for half an hour. RESULTS: The protein expressions of LC3-II and Atg5 appeared immediately after UV exposure and then vanished 1 h later. The autophagosome formation in coelomic fluid cells confirmed the autophagy appearance pattern of LC3-II and Atg5. The sea cucumber individuals maintained the health condition during the entire event of autophagy. The autophagic inhibitor along with UV exposure contributed to sea cucumber's swollen intestinal tissues, but the autophagic inducer functioned to alleviate and neutralize the UV effect. CONCLUSIONS: The autophagy procedure analysis demonstrated that autophagy plays a role to maintain the health condition of sea cucumber during autolysis inducement. The autolysis of sea cucumber can be alleviated or postponed by the exogenous autophagy inducer and this finding would benefit the live sea cucumber transportation. © 2021 Society of Chemical Industry.

Análise de algoritmo diagnóstico de febre amarela em amostras de primatas não humanos encaminhadas ao Centro de Patologia do Instituto Adolfo Lutz / Analysis of a diagnostic algorithm for yellow fever in primate samples non-humans sent to the Pathology Center of Instituto Adolfo Lutz

O Centro de Patologia do Instituto Adolfo Lutz (CPA-IAL) é credenciado pelo Ministério da Saúde como laboratório de referência macrorregional para a vigilância epidemiológica de febre amarela (FA) em seres humanos e primatas não humanos (PNH) do Brasil, atuando por meio de análise histopatológica e imuno-histoquímica (IHQ). Até o ano de 2018, ambos os exames eram aplicados a todas as amostras de PNH recebidas para a pesquisa de FA. Em 2019, implantou-se um algoritmo diagnóstico baseado na triagem pelas características histopatológicas observadas no tecido hepático, possibilitando a racionalização do uso da IHQ. Objetivo: Avaliar a aplicação do algoritmo diagnóstico comparado ao período que antecedeu sua implantação. Métodos: Estudo retrospectivo de relatórios anatomopatológicos de PNH emitidos, entre 2018 e 2019, no CPA-IAL para determinação de índices de performance diagnóstica do exame histopatológico na vigilância epidemiológica de febre amarela, avaliação da sensibilidade do exame imuno-histoquímico para amostras com autólise de moderada a avançada e comparação da mediana de tempo decorrido para emissão dos relatórios em cada período. Resultados: Não houve diferença estatisticamente significante na performance da detecção de FA por histologia e IHQ entre os períodos pré e pós algoritmo; houve importante redução na quantidade de exames IHQ solicitados e no tempo de liberação dos relatórios (p<0,0001). Conclusões: O algoritmo resultou em desempenho semelhante, redução do tempo de liberação oportuno para a vigilância epidemiológica do agravo e da quantidade de reações IHQ realizadas, portanto, apresentando-se adequado para o diagnóstico de febre amarela em PNH no CPA-IAL.

Temporal Sequence of Post-Mortem Autolysis in the Mouse Retina.

In order to better differentiate ante-mortem lesions from post-mortem retinal autolysis, the temporal sequence of post-mortem changes was studied in a well-controlled mouse model. Mice were of the same strain, age and sex, and were held at a constant ambient temperature. Eyes were collected at various times up to 72 h after death and immersion-fixed in either Davidson's fixative or 10% neutral buffered formalin, paraffin-embedded and sections cut and stained with haematoxylin and eosin. The most prominent, and early, autolytic change was retinal detachment, and subsequent folding, which occurred immediately after death in formalin-fixed eyes, but not until 2 h post mortem with Davidson's fixative. Retinal separation was complete at 16 h, or almost complete by 2 h, in formalin, but in Davidson's fixative, was only partial and segmental, the latter not becoming total until much later. Retinal detachment was attended by progressively more severe disruption and dissolution of photoreceptors and, particularly in Davidson's-fixed retinas, the rod outer segment often showed marked homogenization from 30 min to 4 h after death. The other major early change was nuclear pyknosis in the inner nuclear layer. Ganglion cells initially had cytoplasmic swelling, followed by shrinkage and basophilia (at 4 h with formalin and 16 h with Davidson's), with nuclear pyknosis becoming increasingly common over time. While the three retinal neuronal layers eventually became more attenuated and depleted of cells, the thickness of these layers was augmented by severe swelling. These findings show that the post-mortem interval at which histological interpretation of retinal changes becomes potentially compromised is dependent on the duration of this interval and the fixative used.

Quantifying Staphylococcus aureus Membrane Potential Using Flow Cytometry.

Quantifying fluorescent markers in cell populations using flow cytometry has been a powerful technological advance. Fluorescent properties of cyanine dyes coupled with flow cytometry allow investigators to monitor the membrane potential (MP), an important component of the proton motive force (PMF). MP (or ΔΨ) is the electrical potential across the cell membrane. The other component of the PMF is ΔpH, or the difference in interior and exterior proton concentrations. MP plays a critical role in bacterial physiology. In Staphylococcus aureus, MP is required for generation of ATP, regulating autolytic activity, maintaining ion homeostasis, and resistance to some classes of antibiotics. This protocol exploits unique spectral and physical properties of the cyanine-based molecule diethyloxacarbocyanine iodide, or DiOC, and flow cytometry technology to quantify MP in S. aureus. This assay has been used by researchers to define the electron transport chain of S. aureus as well as determine how intrinsic and extrinsic factors affect MP.

Innovative biosynthesis of silver nanoparticles using yeast glucan nanopolymer and their potentiality as antibacterial composite.

Nanometals (NM) frequently possess potent antimicrobial potentials to combat various pathogens, but their elevated biotoxicity limits their direct applications. The biosynthesis of NM and their capping/conjugation with natural biopolymers can effectually enhance NM stability and diminish such toxicity. Yeast ß-glucan (ßG), from Saccharomyces cerevisiae, was extracted and transformed to nanoparticles (NPs) using alkali/acid facile protocol. The ßG NPs were innovatively employed for direct biosynthesis of silver nanoparticles (Ag NPs) without extra chemical processes. The physicochemical assessments (Fourier-transform infrared, X-ray diffraction, and transmission electron microscopy) validated NPs formation, interaction, and interior capping of Ag NPs in ßG NPs. The synthesized ßG NPs, Ag NPs, and ßG-Ag NPs composite were negatively charged and had minute particle sizes with mean diameters of 58.65, 6.72, and 63.88 nm, respectively. The NPs (plain Ag NPs and composited ßG-Ag NPs) exhibited potent comparable bactericidal actions, opposing Gram+ (Staphylococcus aureus) and Gram- (Escherichia coli, Salmonella Typhimurium, and Pseudomonas aeruginosa). Scanning micrographs, of treated S. aureus and S. Typhimurium with ßG-Ag NPs, elucidated the powerful bactericidal actions of nanocomposite for destructing pathogens' cells. The inventive Ag NPs biosynthesis with ßG NPs and the combined ßG-Ag NPs nanocomposites could be impressively recommended as powerful antibacterial candidates with minor potential toxicity.

Autolysis-mediated membrane vesicle formation in Bacillus subtilis.

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.

Endolysin-Based Autolytic E. coli System for Facile Recovery of Recombinant Proteins.

Recovery of recombinant proteins from the Escherichia coli cytoplasm depends on cell disruption by mechanical, chemical, and/or enzymatic methods, which usually cause incomplete cell breakage or protein denaturation. Controllable autolytic E. coli strains have been designed to facilitate the purification of recombinant proteins; however, these strains suffer from low recovery yield, slow cell lysis, or extensive strain engineering. Herein, we report an improved, highly efficient programmable autolytic E. coli platform, in which cell lysis is initiated upon the induced expression of T4 lysozyme with N-terminal fusion of a cell-penetrating peptide. Through the engineering of the peptide sequence and copy number, and by incorporating the fusion lytic gene into the E. coli genome, more than 99.97% of cells could be lysed within 30 min of induction regardless of cell age. We further tested the expression and release of a recombinant enzyme lysostaphin (Lst) and demonstrated that 4 h induction of the lytic gene after 3 h of Lst expression resulted in 98.97% cell lysis. Lst obtained from this system had the same yield, yet 1.63-fold higher activity, compared with that obtained from cells lysed by freeze-thawing and sonication. This autolytic platform shows potential for use in large-scale microbial production of proteins and other biopolymers.

Autolysis in Crustacean Tissues after Death: A Case Study Using the Procambarus clarkii Hepatopancreas.

Autolysis is an internal phenomenon following the death of an organism that leads to the degradation of tissues. In order to explore the initial stages of autolysis and attempt to establish reference standards for tissue changes after death, we studied the rapidly autolyzing tissue of the crayfish hepatopancreas. Samples from the hepatopancreas of crayfish were examined 0, 5, 10, 30, 60, and 120 minutes after death. Histological and ultrapathological examinations and evaluations and apoptotic cell counts were conducted to determine the initiation time and degree of autolysis. The results showed that autolysis in the hepatopancreas of crayfish began within 5 minutes. Initially, autolysis manifested in the swelling of hepatic tubular cells and the widening of mesenchyme. Cells undergoing autolysis showed severe organelle necrolysis. Based on these observations, tissue samples should be collected and preserved within five minutes to avoid interfering with histopathological diagnoses.