Robot-Assisted Correction of a Supra-Long Tracheal Stenosis Using C-Type Nickel-Titanium Alloy Exterior Stenting and Suspension Fixation Technique: A Case Report.

T-tubes and airway stents are commonly used but have limited effectiveness and frequent complications. A 50-year-old male patient presented with severe tracheal stenosis, affecting an 8.7 cm length of the airway. We employed an innovative approach known as external suspension fixation of tracheal stent using robotic assistance. This method involves surgically attaching the stent to the exterior of the trachea to provide support and stabilize the softened or collapsed tracheal segments. We designed a C-shaped nickel-titanium alloy exterior stent and successfully fixed it using robotic assistance. This intervention effectively restored tracheal function and led to a favorable postoperative recovery. The technique does not affect tracheal membrane function or airway mucociliary clearance. It could potentially be considered as a new option for treating long-segment benign tracheal softening or collapse.

Transcriptome and Lipidomic Analysis Suggests Lipid Metabolism Reprogramming and Upregulating SPHK1 Promotes Stemness in Pancreatic Ductal Adenocarcinoma Stem-like Cells.

Cancer stem cells (CSCs) are considered to play a key role in the development and progression of pancreatic ductal adenocarcinoma (PDAC). However, little is known about lipid metabolism reprogramming in PDAC CSCs. Here, we assigned stemness indices, which were used to describe and quantify CSCs, to every patient from the Cancer Genome Atlas (TCGA-PAAD) database and observed differences in lipid metabolism between patients with high and low stemness indices. Then, tumor-repopulating cells (TRCs) cultured in soft 3D (three-dimensional) fibrin gels were demonstrated to be an available PDAC cancer stem-like cell (CSLCs) model. Comprehensive transcriptome and lipidomic analysis results suggested that fatty acid metabolism, glycerophospholipid metabolism, and, especially, the sphingolipid metabolism pathway were mostly associated with CSLCs properties. SPHK1 (sphingosine kinases 1), one of the genes involved in sphingolipid metabolism and encoding the key enzyme to catalyze sphingosine to generate S1P (sphingosine-1-phosphate), was identified to be the key gene in promoting the stemness of PDAC. In summary, we explored the characteristics of lipid metabolism both in patients with high stemness indices and in novel CSLCs models, and unraveled a molecular mechanism via which sphingolipid metabolism maintained tumor stemness. These findings may contribute to the development of a strategy for targeting lipid metabolism to inhibit CSCs in PDAC treatment.

Complete Genome Sequence of Bacillus licheniformis Strain GN02, Isolated from the Root Surface of Brassica chinensis.

Bacillus licheniformis GN02 was isolated from the root surface of Pak Choi Cabbage (Brassica chinensis). Here, we report the whole-genome sequence of strain GN02, which includes only a circular chromosome (4,252,022 bp; GC content, 46.08%).

Bacillus paralicheniformis RP01 Enhances the Expression of Growth-Related Genes in Cotton and Promotes Plant Growth by Altering Microbiota inside and outside the Root.

Plant growth-promoting bacteria (PGPB) can promote plant growth in various ways, allowing PGPB to replace chemical fertilizers to avoid environmental pollution. PGPB is also used for bioremediation and in plant pathogen control. The isolation and evaluation of PGPB are essential not only for practical applications, but also for basic research. Currently, the known PGPB strains are limited, and their functions are not fully understood. Therefore, the growth-promoting mechanism needs to be further explored and improved. The Bacillus paralicheniformis RP01 strain with beneficial growth-promoting activity was screened from the root surface of Brassica chinensis using a phosphate-solubilizing medium. RP01 inoculation significantly increased plant root length and brassinosteroid content and upregulated the expression of growth-related genes. Simultaneously, it increased the number of beneficial bacteria that promoted plant growth and reduced the number of detrimental bacteria. The genome annotation findings also revealed that RP01 possesses a variety of growth-promoting mechanisms and a tremendous growth-promoting potential. This study isolated a highly potential PGPB and elucidated its possible direct and indirect growth-promoting mechanisms. Our study results will help enrich the PGPB library and provide a reference for plant-microbe interactions.

Shear wave elastography of the diaphragm in acute exacerbation of chronic obstructive pulmonary disease: A prospective observational study.

Patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) are prone to diaphragmatic dysfunction. However, dynamic assessment of diaphragmatic function is complex and difficult, and whether the assessment of diaphragmatic function can reflect clinical indicators such as lung function in AECOPD patients remains unclear. We studied diaphragm stiffness and diaphragm stiffening rate (DSR) in AECOPD patients with acute exacerbations ≥ 2 times within 1 year and their correlation with clinical data, the diaphragmatic thickening fraction (DTF), lung function, and blood gas values. In total, 112 AECOPD patients in group C and Group D who had acute exacerbations ≥ 2 times within 1 year in the Global Initiative for Chronic Obstructive Lung Disease Guideline A (low risk, few symptoms), B (low risk, many symptoms), C (High risk, few symptoms), D (High risk, many symptoms) grouping system were included in the study. Their general clinical data, chronic obstructive pulmonary disease assessment test (CAT), modified medical research council (mMRC), number of acute exacerbations in 1 year, DTF, lung function, and blood gas analysis were collected. The diaphragm shear wave elasticity at functional residual capacity (DsweFRC) and DSR were measured by ultrasound. The DsweFRC and DSR of Group D were higher than those of Group C (P < .05). DsweFRC, DSR were negatively correlated with DTF, forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC) and FEV1/FVC (r ranged from -0.293 to -0.697, all P < .05), and positively correlated with CAT score, mMRC score, and arterial carbon dioxide pressure (r ranged from 0.274 to 0.462, all P < .05) in both groups; the correlation coefficients of DsweFRC, DSR and DTF, FEV1/FVC in group D were greater than those in group C. There was no correlation between DsweFRC, DSR and arterial oxygen partial pressure in both groups (P > .05). The DsweFRC, DSR increased with the number of acute exacerbations per year in both groups. We found that diaphragmatic stiffness in AECOPD patients increased with the number of acute exacerbations within 1 year, correlated with DTF, CAT, mMRC, lung function, and arterial carbon dioxide pressure and provides a simple and practical method for dynamically assessing diaphragmatic function and disease severity in AECOPD patients.

Robot-assisted resection of multiple lung nodules through combination of intercostal incisions and a subxiphoid incision as a utility port.

INTRODUCTION: Robotic-assisted thoracic surgery (RATS) via subxiphoid incision may be superior in resection of multiple lung nodules. METHODS: Outcomes of robot-assisted one-stage bilateral lobectomy or segmentectomy via intercostal and subxiphoid incisions for multiple ground-glass opacities were analysed. RESULTS: Total 36 cases were analysed in this study. Thirteen cases had bilateral lobectomy + segmentectomy, 15 cases underwent bilateral segmentectomies, and 8 cases underwent lobectomy + segmentectomy + wedge resection. The average intraoperative blood loss was 110.2 ± 57.8 mL, operation time was 154 ± 64.2 min, thoracic draining time was 2.6 ± 3.2 days, and postoperative hospital stay was 4.8 ± 3.3 days. Three patients had atrial fibrillation and 3 patients had continuous air leakage for over 3 days, but there was no death or postoperative pain. CONCLUSION: Robot-assisted one-stage bilateral resection of multiple lung nodules through combination of intercostal and subxiphoid incision as a utility port is safe and reliable.

Tumor-associated macrophages-educated reparative macrophages promote diabetic wound healing.

Nonhealing diabetic wounds, with persistent inflammation and damaged vasculature, have failed conventional treatments and require comprehensive interference. Here, inspired by tumor-associated macrophages (TAMs) that produce abundant immunosuppressive and proliferative factors in tumor development, we generate macrophages to recapitulate TAMs' reparative functions, by culturing normal macrophages with TAMs' conditional medium (TAMs-CM). These TAMs-educated macrophages (TAMEMs) outperform major macrophage phenotypes (M0, M1, or M2) in suppressing inflammation, stimulating angiogenesis, and activating fibroblasts in vitro. When delivered to skin wounds in diabetic mice, TAMEMs efficiently promote healing. Based on TAMs-CM's composition, we further reconstitute a nine-factor cocktail to train human primary monocytes into TAMEMsC-h , which fully resemble TAMEMs' functions without using tumor components, thereby having increased safety and enabling the preparation of autologous cells. Our study demonstrates that recapitulating TAMs' unique reparative activities in nontumor cells can lead to an effective cell therapeutic approach with high translational potential for regenerative medicine.

Characterizing the Proliferation Patterns of Representative Microsporidian Species Enlightens Future Studies of Infection Mechanisms.

BACKGROUND: Microsporidia are a group of pathogens that infect all kinds of animals, such as humans, silkworms, honeybees, and shrimp; they, therefore, pose a severe threat to public health and the economy. There are over 1500 species of microsporidia that have been reported, among which Encephalitozoon hellem and Nosema bombycis are the representative zoonotic and insect-infecting species, respectively. Investigating their cell infection patterns is of great significance for understanding their infection mechanisms. METHODS: Specific probes were designed for the ribosomal RNA sequences of microsporidia. Fluorescence in situ hybridization (FISH) was used to trace the proliferation cycle of the pathogens in different cells. RESULTS: Here, two rRNA large subunit gene (LSUrRNA) probes specifically labeling N. bombycis were obtained. The life cycle of N. bombycis in silkworm cells and E. hellem in three kinds of host cells was graphically drawn. N. bombycis meronts were first observed at 30 hours post-infection (hpi), and they began merogony. Sporonts were observed at 42 hpi, and the first entire proliferation cycle was completed at 48 hpi. The proliferation cycle of E. hellem in RK13 and HEK293 epithelial cells was almost the same, completing the first life cycle after 24 hpi, but it was significantly delayed to 32 hpi in RAW264.7. CONCLUSIONS: Specific FISH probes were established for labeling microsporidia in multiple host cells. The proliferation characteristics of representative zoonotic and insect-infecting microsporidian species were clarified. This study provides an experimental pattern for future analyses of microsporidian infection mechanisms.

Discovery of MAO-B Inhibitor with Machine Learning, Topomer CoMFA, Molecular Docking and Multi-Spectroscopy Approaches.

Alzheimer's disease (AD) is the most common type of dementia and is a serious disruption to normal life. Monoamine oxidase-B (MAO-B) is an important target for the treatment of AD. In this study, machine learning approaches were applied to investigate the identification model of MAO-B inhibitors. The results showed that the identification model for MAO-B inhibitors with　K-nearest neighbor(KNN) algorithm had a prediction accuracy of 94.1% and 88.0% for the 10-fold cross-validation test and the independent test set, respectively. Secondly, a quantitative activity prediction model for MAO-B was investigated with the Topomer CoMFA model. Two separate cutting mode approaches were used to predict the activity of MAO-B inhibitors. The results showed that the cut model with q2 = 0.612 (cross-validated correlation coefficient) and r2 = 0.824 (non-cross-validated correlation coefficient) were determined for the training and test sets, respectively. In addition, molecular docking was employed to analyze the interaction between MAO-B and inhibitors. Finally, based on our proposed prediction model, 1-(4-hydroxyphenyl)-3-(2,4,6-trimethoxyphenyl)propan-1-one (LB) was predicted as a potential MAO-B inhibitor and was validated by a multi-spectroscopic approach including fluorescence spectra and ultraviolet spectrophotometry.

Radiomics combined with clinical features in distinguishing non-calcifying tuberculosis granuloma and lung adenocarcinoma in small pulmonary nodules.

Aim: To evaluate the performance of radiomics models with the combination of clinical features in distinguishing non-calcified tuberculosis granuloma (TBG) and lung adenocarcinoma (LAC) in small pulmonary nodules. Methodology: We conducted a retrospective analysis of 280 patients with pulmonary nodules confirmed by surgical biopsy from January 2017 to December 2020. Samples were divided into LAC group (n = 143) and TBG group (n = 137). We assigned them to a training dataset (n = 196) and a testing dataset (n = 84). Clinical features including gender, age, smoking, CT appearance (size, location, spiculated sign, lobulated shape, vessel convergence, and pleural indentation) were extracted and included in the radiomics models. 3D slicer and FAE software were used to delineate the Region of Interest (ROI) and extract clinical features. The performance of the model was evaluated by the Area Under the Receiver Operating Characteristic (ROC) Curve (AUC). Results: Based on the model selection, clinical features gender, and age in the LAC group and TBG group showed a significant difference in both datasets (P < 0.05). CT appearance lobulated shape was also significantly different in the LAC group and TBG group (Training dataset, P = 0.034; Testing dataset, P = 0.030). AUC were 0.8344 (95% CI [0.7712-0.8872]) and 0.751 (95% CI [0.6382-0.8531]) in training and testing dataset, respectively. Conclusion: With the capacity to detect differences between TBG and LAC based on their clinical features, radiomics models with a combined of clinical features may function as the potential non-invasive tool for distinguishing TBG and LAC in small pulmonary nodules.

Loss of GABARAPL1 confers ferroptosis resistance to cancer stem-like cells in hepatocellular carcinoma.

Cancer stem-like cells (CSLC) are considered a major contributor to the development and progression of hepatocellular carcinoma (HCC). Previous studies indicated that CSLC are characterized by resistance to ferroptosis, a type of lipid peroxidation-dependent cell death. Here, we identified a set of ferroptosis-related stemness genes (FRSG) and found that these genes may be involved in immune infiltration in HCC. A four-FRSG (CDKN2A, GABARAPL1, HRAS, RPL8) risk model with prognostic prediction was constructed by a Cox analysis in HCC. Among these four genes, GABARAPL1 was downregulated in HCC tumor-repopulating cells (TRC; a type of CSLC). Its downregulation decreased the sensitivity of HCC TRC to erastin- or sorafenib-triggered ferroptosis. Together, we uncovered a molecular mechanism via which CSLC could achieve tolerance to ferroptosis. Further studies may provide potential therapeutic strategies targeting CSLC in HCC.

Microsporidia Promote Host Mitochondrial Fragmentation by Modulating DRP1 Phosphorylation.

Microsporidia are obligate intracellular parasites that infect a wide variety of hosts ranging from invertebrates to vertebrates. These parasites have evolved strategies to directly hijack host mitochondria for manipulating host metabolism and immunity. However, the mechanism of microsporidia interacting with host mitochondria is unclear. In the present study, we show that microsporidian Encephalitozoon greatly induce host mitochondrial fragmentation (HMF) in multiple cells. We then reveal that the parasites promote the phosphorylation of dynamin 1-like protein (DRP1) at the 616th serine (Ser616), and dephosphorylation of the 637th serine (Ser637) by highly activating mitochondrial phosphoglycerate mutase 5 (PGAM5). These phosphorylation modifications result in the translocation of DRP1 from cytosol to the mitochondrial outer membrane, and finally lead to HMF. Furthermore, treatment with mitochondrial division inhibitor 1 (Mdivi1) significantly reduced microsporidian proliferation, indicating that the HMF are crucial for microsporidian replication. In summary, our findings reveal the mechanism that microsporidia manipulate HMF and provide references for further understanding the interactions between these ubiquitous pathogens with host mitochondria.

Characterization of the Largest Secretory Protein Family, Ricin B Lectin-like Protein, in Nosema bombycis: Insights into Microsporidian Adaptation to Host.

Microsporidia are a group of obligate intracellular pathogens infecting nearly all animal phyla. The microsporidian Nosema bombycis has been isolated from several lepidopteran species, including the economy-important silkworms as well as several crop pests. Proteins secreted by parasites can be important virulent factors in modulating host pathways. Ricin is a two-chain lectin best known for its extreme vertebrate toxicity. Ricin B lectin-like proteins are widely distributed in microsporidia, especially in N. bombycis. In this study, we identify 52 Ricin B lectin-like proteins (RBLs) in N. bombycis. We show that the N. bombycis RBLs (NbRBLs) are classified into four subfamilies. The subfamily 1 was the most conserved, with all members having a Ricin B lectin domain and most members containing a signal peptide. The other three subfamilies were less conserved, and even lost the Ricin B lectin domain, suggesting that NbRBLs might be a multi-functional family. Our study here indicated that the NbRBL family had evolved by producing tandem duplications firstly and then expanded by segmental duplications, resulting in concentrated localizations mainly in three genomic regions. Moreover, based on RNA-seq data, we found that several Nbrbls were highly expressed during infection. Further, the results show that the NbRBL28 was secreted into host nucleus, where it promotes the expressions of genes involved in cell cycle progression. In summary, the great copy number, high divergence, and concentrated genome distribution of the NbRBLs demonstrated that these proteins might be adaptively evolved and played a vital role in the multi-host N. bombycis.

Glucose oxidase loaded Cu2+ based metal-organic framework for glutathione depletion/reactive oxygen species elevation enhanced chemotherapy.

INTRODUCTION: The development of multidrug resistance (MDR) is a major cause for the failure of chemotherapy, which requires the aid of nanomedicine. METHODS: Here in our study, a Cu2+ based metal-organic framework (COF) was firstly developed and employed as a carrier for the delivery of glucose oxidase (GOx) and doxorubicin (Dox) (COF/GOx/Dox) for the therapy of MDR lung cancers. RESULTS: Our results showed that the GOx can catalyze glucose and produce H2O2. In the mean time, the Cu2+ can react with GSH and then transform into Cu+, which resulted in GSH depletion. Afterwards, the produced Cu+ and H2O2 trigger Fenton reaction to generate ROS to damage the redox equilibrium of cancer cells. Both effects contributed to the reverse of MDR in A549/Dox cells and finally resulted in significantly enhanced in vitro/in vivo anticancer performance. DISCUSSION: The combination of glutathione depletion/reactive oxygen species elevation might be a promising strategy to enhance the efficacy of chemotherapy and reverse MDR in cancers.

Proteomic Identification of Bombyx mori Organelles Using the Engineered Ascorbate Peroxidase APEX and Development of Silkworm Organelle Proteome Database (SilkOrganPDB).

Silkworm Bombyx mori is an economically important insect and a lepidopteran model. Organelle proteome is vital to understanding gene functions; however, it remains to be identified in silkworm. Here, using the engineered ascorbate peroxidase APEX, we constructed transgenic B. mori embryo cells (BmE) expressing APEX-NLS, COX4-APEX, APEX-Rev, and APEX-KDEL in nucleus, mitochondrial matrix (MM), cytosol, and endoplasmic reticulum (ER), and isolated the biotin-labeled proteins using streptavidin-affinity purification, respectively. The isolated proteins were determined using LC-MS/MS and annotated by searching B. mori genomes downloaded from GenBank, SilkBase, SilkDB 2.0, and SilkDB 3.0, resulting in 842, 495, 311, and 445 organelle proteins identified, respectively. We mapped the 296 MM proteins annotated in the GenBank data to mitochondrial protein databases of the fly, human, and mouse, and found that 140 (47%) proteins are homologous to 80 fly proteins, and 65 (22%) proteins match to 31 and 29 human and mouse proteins, respectively. Protein orthology was predicted in multiple insects using OrthoMCL, producing 460 families containing 839 proteins we identified. Out of 460 families, 363 were highly conserved and found in all insects, leaving only three proteins without orthology in other insects, indicating that the identified proteins are highly conserved and probably play important roles in insects. A gene ontology enrichment analysis by clusterProfiler revealed that the nucleus proteins significantly enriched in cellular component terms of nucleus and nucleolus, the MM proteins markedly enriched in molecular function terms of nucleotide binding, and the cytosol proteins mainly enriched in biological process terms of small molecule metabolism. To facilitate the usage and analysis of our data, we developed an open-access database, Silkworm Organelle Proteome Database (SilkOrganPDB), which provides multiple modules for searching, browsing, downloading, and analyzing these proteins, including BLAST, HMMER, Organelle Proteins, Protein Locations, Sequences, Gene Ontology, Homologs, and Phylogeny. In summary, our work revealed the protein composition of silkworm BmE organelles and provided a database resource helpful for understanding the functions and evolution of these proteins.

Microsporidia infection upregulates host energy metabolism but maintains ATP homeostasis.

Microsporidia are a group of obligate intracellular parasites which lack mitochondria and have highly reduced genomes. Therefore, they are unable to produce ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Instead, they have evolved strategies to obtain and manipulate host metabolism to acquire nutrients. However, little is known about how microsporidia modulate host energy metabolisms. Here, we present the first targeted metabolomics study to investigate changes in host energy metabolism as a result of infection by a microsporidian. Metabolites of silkworm embryo cell (BmE) were measured 48 h post infection by Nosema bombycis. Thirty metabolites were detected, nine of which were upregulated and mainly involved in glycolysis (glucose 6-phosphate, fructose 1,6-bisphosphate) and the TCA cycle (succinate, α-ketoglutarate, cis-aconitate, isocitrate, citrate, fumarate). Pathway enrichment analysis suggested that the upregulated metabolites could promote the synthesization of nucleotides, fatty acids, and amino acids by the host. ATP concentration in host cells, however, was not significantly changed by the infection. This ATP homeostasis was also found in Encephalitozoon hellem infected mouse macrophage RAW264.7, human monocytic leukemia THP-1, human embryonic kidney 293, and human foreskin fibroblast cells. These findings suggest that microsporidia have evolved strategies to maintain levels of ATP in the host while stimulating metabolic pathways to provide additional nutrients for the parasite.

Expression and Identification of a Novel Spore Wall Protein in Microsporidian Nosema bombycis.

Microsporidia are a group of obligate intracellular parasites causing significant disease in human beings and economically important animals. Though a few spore wall proteins (SWPs) have now been identified in these intriguing species, the information on SWPs remains too little to elucidate the spore wall formation mechanisms of microsporidia. It has been well described that numerous proteins with tandem repeats tend to be localized on the cell wall of fungi and parasites. Previously, by scanning the proteins with tandem repeats in microsporidian Nosema bombycis, we obtained 83 candidate SWPs based on whether those proteins possess a signal peptide and/or transmembrane domain. Here, we further characterized a candidate protein (EOB13250) with three tandem repeats in the N-terminal region and a transmembrane domain in C-terminus of N. bombycis. Sequence analysis showed that the tandem repeat domain of EOB13250 was species-specific for this parasite. RT-PCR indicated that the expression of the gene encoding this protein started on the fourth day postinfection. After cloned and expressed in Escherichia coli, a polyclone antibody against the recombinant EOB13250 protein was prepared. Western blotting demonstrated this protein exist in N. bombycis. Immunofluorescence analysis (IFA) and immunoelectron microscopy analysis (IEM) further provided evidence that EOB13250 was an endospore wall protein. These results together suggested that EOB13250 was a novel spore wall protein of N. bombycis. This study provides a further enrichment of the number of identified spore wall proteins in microsporidia and advances our understanding of the spore wall formation mechanism in these obligate unicellular parasites.

Innate and Adaptive Immune Responses Against Microsporidia Infection in Mammals.

Microsporidia are obligate intracellular and eukaryotic pathogens that can infect immunocompromised and immunocompetent mammals, including humans. Both innate and adaptive immune systems play important roles against microsporidian infection. The innate immune system can partially eliminate the infection by immune cells, such as gamma delta T cell, natural killer cells (NKs), macrophages and dendritic cells (DCs), and present the pathogens to lymphocytes. The innate immune cells can also prime and enhance the adaptive immune response via surface molecules and secreted cytokines. The adaptive immune system is critical to eliminate microsporidian infection by activating cytotoxic T lymphocyte (CTL) and humoral immune responses, and feedback regulation of the innate immune mechanism. In this review, we will discuss the cellular and molecular responses and functions of innate and adaptive immune systems against microsporidian infection.

In-vitro cultivation of Nosema bombycis sporoplasms: A method for potential genetic engineering of microsporidia.

Microsporidia are obligate intracellular parasites and cannot be cultured in vitro, which limits the use of current genetic engineering technologies on this pathogen. We isolated sporoplasms of Nosema bombycis to attempt to culture the pathogen in vitro. Cell-free medium was designed and successfully maintained the sporoplasms for 5 days. The sporoplasms were able to absorb ATP from the medium and DNA replicated during cultivation, although there was not a significant change in morphology and number of sporoplasms. Our study provides a strategy for in vitro cultivation and genetic manipulation of microsporidia. .

Characterization of Hsp70 gene family provides insight into its functions related to microsporidian proliferation.

Heat shock protein 70 (Hsp70), a highly conserved protein family, is widely distributed in organisms and plays fundamental roles in biotic and abiotic stress responses. However, reports on Hsp70 genes are scarce in microsporidia, a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. In this study, we identified 37 Hsp70 proteins from eight microsporidian genomes and classified them into four subfamilies (A-D). The number of Hsp70 genes in these microsporidia was significantly fewer than in Rozella allomycis and yeast. All microsporidian species contained genes from each subfamily and similar subcellular locations (mitochondria, endoplasmic reticulum, cytosol, and cytosol and/or nucleus), indicating that each Hsp70 member may fulfil distinct functions. The conserved structures and motifs of the Hsp70 proteins in the same subfamily were highly similar. Expression analysis indicated that the subfamily C cytosol (cyto)-associated Hsp70s is functional during microsporidia development. Immunofluorescence assays revealed that Cyto-NbHsp70 was cytoplasmically located in the proliferation-stage of Nosema bombycis. Cyto-NbHsp70 antiserum also labeled Encephalitozoon hellem within infected cells, suggesting that this antiserum is a potential molecular marker for labeling the proliferative phases of different microsporidian species. The propagation of N. bombycis was significantly inhibited following RNAi of Cyto-NbHsp70, indicating that Cyto-NbHsp70 is important for pathogen proliferation. Our phylogenetic data suggest that Hsp70 proteins evolved during microsporidia adaption to intracellular parasitism, and they play important roles in pathogen development.