Transanal minimally invasive surgery (TAMIS) for local excision of benign and malignant rectal neoplasia: a 7-year experience.

PURPOSE: Transanal minimally invasive surgery (TAMIS) is an advanced transanal platform that can be utilised to perform high-quality local excision (LE) of rectal neoplasia. This study describes clinical and midterm oncological outcomes from a single unit's 7-year experience with TAMIS. METHODS: Consecutive patients who underwent TAMIS LE at our institution between January 1st, 2016, and December 31st, 2022, were identified from a prospectively maintained database. Indication for TAMIS LE was benign lesions not amenable to endoscopic excision or histologically favourable early rectal cancers. The primary endpoints were resection quality, disease recurrence and peri-operative outcomes. The Kaplan-Meier survival analyses were used to describe disease-free survival (DFS) for patients with rectal adenocarcinoma that did not receive immediate salvage proctectomy. RESULTS: There were 168 elective TAMIS LE procedures performed for 102 benign and 66 malignant lesions. Overall, a 95.2% negative margin rate was observed, and 96.4% of lesions were submitted without fragmentation. Post-operative morbidity was recorded in 8.3% of patients, with post-operative haemorrhage, being the most common complication encountered. The mean follow-up was 17 months (SD 15). Local recurrence occurred in 1.6%, and distant organ metastasis was noted in 1.6% of patients. CONCLUSIONS: For carefully selected patients, TAMIS for local excision of early rectal neoplasia is a valid option with low morbidity that maintains the advantages of organ preservation.

Adjuvanted Fusion Protein Vaccine Induces Durable Immunity to Onchocerca volvulus in Mice and Non-Human Primates.

Onchocerciasis remains a debilitating neglected tropical disease. Due to the many challenges of current control methods, an effective vaccine against the causative agent Onchocerca volvulus is urgently needed. Mice and cynomolgus macaque non-human primates (NHPs) were immunized with a vaccine consisting of a fusion of two O. volvulus protein antigens, Ov-103 and Ov-RAL-2 (Ov-FUS-1), and three different adjuvants: Advax-CpG, alum, and AlT4. All vaccine formulations induced high antigen-specific IgG titers in both mice and NHPs. Challenging mice with O. volvulus L3 contained within subcutaneous diffusion chambers demonstrated that Ov-FUS-1/Advax-CpG-immunized animals developed protective immunity, durable for at least 11 weeks. Passive transfer of sera, collected at several time points, from both mice and NHPs immunized with Ov-FUS-1/Advax-CpG transferred protection to naïve mice. These results demonstrate that Ov-FUS-1 with the adjuvant Advax-CpG induces durable protective immunity against O. volvulus in mice and NHPs that is mediated by vaccine-induced humoral factors.

Spatiotemporal patterns of wheat response to Pyrenophora tritici-repentis in asymptomatic regions revealed by transcriptomic and X-ray fluorescence microscopy analyses.

Pathogen attacks elicit dynamic and widespread molecular responses in plants. While our understanding of plant responses has advanced considerably, little is known of the molecular responses in the asymptomatic 'green' regions adjoining lesions. Here, we explore gene expression data and high-resolution elemental imaging to report the spatiotemporal changes in the asymptomatic green region of susceptible and moderately resistant wheat cultivars infected with a necrotrophic fungal pathogen, Pyrenophora tritici-repentis. We show, with improved spatiotemporal resolution, that calcium oscillations are modified in the susceptible cultivar, resulting in 'frozen' host defence signals at the mature disease stage, and silencing of the host's recognition and defence mechanisms that would otherwise protect it from further attacks. In contrast, calcium accumulation and a heightened defence response were observed in the moderately resistant cultivar in the later stage of disease development. Furthermore, in the susceptible interaction, the asymptomatic green region was unable to recover after disease disruption. Our targeted sampling technique also enabled detection of eight previously predicted proteinaceous effectors in addition to the known ToxA effector. Collectively, our results highlight the benefits of spatially resolved molecular analysis and nutrient mapping to provide high-resolution spatiotemporal snapshots of host-pathogen interactions, paving the way for disentangling complex disease interactions in plants.

POT-3 preferentially binds the terminal DNA-repeat on the telomeric G-overhang.

Eukaryotic chromosomes typically end in 3' telomeric overhangs. The safeguarding of telomeric single-stranded DNA overhangs is carried out by factors related to the protection of telomeres 1 (POT1) protein in humans. Of the three POT1-like proteins in Caenorhabditis elegans, POT-3 was the only member thought to not play a role at telomeres. Here, we provide evidence that POT-3 is a bona fide telomere-binding protein. Using a new loss-of-function mutant, we show that the absence of POT-3 causes telomere lengthening and increased levels of telomeric C-circles. We find that POT-3 directly binds the telomeric G-strand in vitro and map its minimal DNA binding site to the six-nucleotide motif, GCTTAG. We further show that the closely related POT-2 protein binds the same motif, but that POT-3 shows higher sequence selectivity. Crucially, in contrast to POT-2, POT-3 prefers binding sites immediately adjacent to the 3' end of DNA. These differences are significant as genetic analyses reveal that pot-2 and pot-3 do not function redundantly with each other in vivo. Our work highlights the rapid evolution and specialisation of telomere binding proteins and places POT-3 in a unique position to influence activities that control telomere length.

Immunization with a tri-antigen syphilis vaccine significantly attenuates chancre development, reduces bacterial load, and inhibits dissemination of Treponema pallidum.

Syphilis continues to be a significant public health concern worldwide. The disease is endemic in many low- and middle-income countries, and rates have risen sharply in high-income countries over the last decade. The continued prevalence of infectious and congenital syphilis worldwide highlights the need for the development of an effective syphilis vaccine to complement public health measures for syphilis control. The complex, multi-stage course of syphilis infection necessitates a holistic approach to the development of an effective vaccine, in which immunization prevents both the localized stage of infection (typified by the highly infectious chancre) and the disseminated stages of infection (typified by the secondary rash, neurosyphilis, and destructive tertiary lesions, as well as congenital syphilis). Inhibiting development of the infectious chancre would reduce transmission thus providing community- level protection, while preventing dissemination would provide individual-level protection by reducing serious sequelae and may also provide community level protection by reducing shedding during secondary syphilis. In the current study we build upon prior investigations which demonstrated that immunizations with individual, well characterized T. pallidum TprK, TprC, and Tp0751 peptides elicits partial protection against infection in the animal model. Specifically, we show here that immunization with a TprC/TprK/Tp0751 tri-antigen cocktail protects animals from progressive syphilis lesions and substantially inhibits dissemination of the infection.

Racial and social disparities in Ventura County, California related to agricultural pesticide applications and toxicity.

Application of agricultural pesticides poses health concerns for farmworkers and for local communities due to pesticide drift from spraying or fumigation, pesticide volatilization into the air, contamination of household dust, as well as direct exposure for people who work in agriculture and their families. In this analysis of pesticide use records for Ventura County, California (USA) from 2016 to 2018, we identified the most prevalent toxicological effects of the pesticides applied. We also developed a cumulative toxicity index that incorporates specific toxicity endpoints for individual pesticides, the severity and strength of association for each endpoint, and the reliability of the data sources. Combining the toxicity index for each pesticide with the pounds applied within each square mile section in Ventura County, we calculated the total toxicity-weighted pesticide use and identified pesticides associated with higher potential risk to health. Analysis of U.S. Census data for Ventura County found a greater percentage of Hispanic/Latino, African American and Asian community members in township sections with a greater volume of pesticides applied and higher toxicity-weighted pesticide use. Similarly, areas with limited economic and social resources had elevated pesticide application overall and elevated toxicity-weighted pesticide use. The combination of toxicological and demographic analyses presented in this study provides information that can support the development of policies to protect public health from excessive exposure to pesticides and better environmental health protection for socially vulnerable populations.

Laboratory testing of sunscreens on the US market finds lower in vitro SPF values than on labels and even less UVA protection.

BACKGROUND: New research has attributed increased significance to the causal link between ultraviolet A (UVA) radiation and immunosuppression and carcinogenesis. In the United States, sunscreens are labeled with only their sun protection factor (SPF) and an imprecise term "broad-spectrum protection." Sunscreen marketing and efficacy evaluations continue to be based primarily on skin redness (sunburn) or erythema. We sought to evaluate the ultraviolet (UV) protection offered by common sunscreen products on the US market using laboratory-measured UV-absorption testing and comparing with computer-modeled protection and the labeled SPF values. This approach enables an investigation of the relationship between the labeled SPF and measured UVA protection, a factor that is ignored in current regulations. METHODS: Fifty-one sunscreen products for sale in the United States with SPF values from 15 to 110 and labeled as providing broad-spectrum protection were tested using a commercial laboratory. All products were evaluated using the ISO 24443:2012 method for sunscreen effectiveness. The final absorbance spectra were used for analysis of in vitro UV protection. RESULTS: In vitro SPF values from laboratory-measured UV absorption and computer modeling were on average just 59 and 42 percent of the labeled SPF. The majority of products provided significantly lower UVA protection with the average unweighted UVA protection factor just 24 percent of the labeled SPF. CONCLUSION: Regulations and marketplace forces promote sunscreens that reduce sunburn instead of products that provide better, more broad-spectrum UV protection. The production and use of products with broad spectrum UV protection should be incentivized, removing the emphasis on sunburn protection and ending testing on people.

Development of a recombinant vaccine against human onchocerciasis.

INTRODUCTION: Human onchocerciasis caused by the filarial nematode parasite Onchocerca volvulus remains a major cause of debilitating disease infecting millions primarily in Sub-Saharan Africa. The development of a prophylactic vaccine, along with mass drug administration, would facilitate meeting the goal of onchocerciasis elimination by 2030. AREAS COVERED: Models used to study immunity to Onchocerca include natural infection of cattle with Onchocerca ochengi and O. volvulus infective third-stage larvae implanted within diffusion chambers in mice. A vaccine, comprised of two adjuvanted recombinant antigens, induced protective immunity in genetically diverse mice suggesting that it will function similarly in diverse human populations. These antigens were recognized by immune humans and also induced protective immunity against Brugia malayi. We describe the development of a fusion protein composed of the two vaccine antigens with the plan to test the vaccine in cows and non-human primates as a prelude to the initiation of phase 1 clinical trials. EXPERT OPINION: The adjuvanted O. volvulus vaccine composed of two antigens Ov-103 and Ov-RAL-2 was shown to be consistently effective at inducing protective immunity using multiple immune mechanisms. The vaccine is ready for further evaluation in other animal models before moving to clinical trials in humans.

Packaged Foods Labeled as Organic Have a More Healthful Profile Than Their Conventional Counterparts, According to Analysis of Products Sold in the U.S. in 2019-2020.

The organic food market's recent rapid global growth reflects the public's interest in buying certified organic foods, including packaged products. Our analysis shows that packaged foods containing fewer ingredients associated with negative public health outcomes are more likely to be labeled organic. Previous studies comparing organic and conventional foods focused primarily on nutrient composition. We expanded this research by additionally examining ingredient characteristics, including processing and functional use. Our dataset included nutrition and ingredient data for 8240 organic and 72,205 conventional food products sold in the U.S. from 2019 to 2020. Compared to conventional foods, organic foods in this dataset had lower total sugar, added sugar, saturated fat and sodium content. Using a mixed effects logistic regression, we found that likelihood of classification as organic increased as sodium content, added sugar content and the number of ultra-processed ingredients and cosmetic additives on the product label decreased. Products containing no trans-fat ingredients were more likely to be labeled organic. A product was more likely to be classified "organic" the more potassium it contained. These features of organic foods sold in the U.S. are significant because lower dietary ingestion of ultra-processed foods, added sugar, sodium and trans-fats is associated with improved public health outcomes.

Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food.

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

Fifteen Years of Sm-p80-Based Vaccine Trials in Nonhuman Primates: Antibodies From Vaccinated Baboons Confer Protection in vivo and in vitro From Schistosoma mansoni and Identification of Putative Correlative Markers of Protection.

Recent advances in systems biology have shifted vaccine development from a largely trial-and-error approach to an approach that promote rational design through the search for immune signatures and predictive correlates of protection. These advances will doubtlessly accelerate the development of a vaccine for schistosomiasis, a neglected tropical disease that currently affects over 250 million people. For over 15 years and with contributions of over 120 people, we have endeavored to test and optimize Sm-p80-based vaccines in the non-human primate model of schistosomiasis. Using RNA-sequencing on eight different Sm-p80-based vaccine strategies, we sought to elucidate immune signatures correlated with experimental protective efficacy. Furthermore, we aimed to explore the role of antibodies through in vivo passive transfer of IgG obtained from immunized baboons and in vitro killing of schistosomula using Sm-p80-specific antibodies. We report that passive transfer of IgG from Sm-p80-immunized baboons led to significant worm burden reduction, egg reduction in liver, and reduced egg hatching percentages from tissues in mice compared to controls. In addition, we observed that sera from Sm-p80-immunized baboons were able to kill a significant percent of schistosomula and that this effect was complement-dependent. While we did not find a universal signature of immunity, the large datasets generated by this study will serve as a substantial resource for further efforts to develop vaccine or therapeutics for schistosomiasis.

Process Development of Sj-p80: A Low-Cost Transmission-Blocking Veterinary Vaccine for Asiatic Schistosomiasis.

Asiatic schistosomiasis caused by Schistosoma japonicum is a neglected tropical disease resulting in significant morbidity to both humans and animals - particularly bovines - in endemic areas. Infection with this parasite leads to less healthy herds, causing problems in communities which rely on bovines for farming, milk and meat production. Additionally, excretion of parasite eggs in feces perpetuates the life cycle and can lead to human infection. We endeavored to develop a minimally purified, inexpensive, and effective vaccine based on the 80 kDa large subunit of the calcium activated neutral protease (calpain) from S. japonicum (Sj-p80). Here we describe the production of veterinary vaccine-grade Sj-p80 at four levels of purity and demonstrate in a pilot study that minimally purified antigen provides protection against infection in mice when paired with a low-cost veterinary adjuvant, Montanide™ ISA61 VG. Preliminary data demonstrate that the vaccine is immunogenic with robust antibody titers following immunization, and vaccination resulted in a reduction of parasite eggs being deposited in the liver (23.4-51.4%) and intestines (1.9-55.1%) depending on antigen purity as well as reducing the ability of these eggs to hatch into miracidia by up to 31.6%. We therefore present Sj-p80 as a candidate vaccine antigen for Asiatic schistosomiasis which is now primed for continued development and testing in bovines in endemic areas. A successful bovine vaccine could play a major role in reducing pathogen transmission to humans by interrupting the parasitic life cycle and improving quality of life for people living in endemic countries.

Structure-based Design of JOC-x, a Conjugatable Tumor Tight Junction Opener to Enhance Cancer Therapy.

Disorganized intercellular junctions are critical for maintaining the integrity of solid epithelial tumors and prevent the infiltration of oncological therapies into the bulk of the malignancy. We have developed small, recombinant proteins which bind a critical junction protein, desmoglein 2, triggering the transient and specific opening of tumor tight junctions allowing for infiltration of the tumor with immune cells, oncolytic viruses, drugs, and other therapeutics. Our new molecule, JOC-x, is a promising candidate for a new class of tumor-targeting agents that accumulate both around and within tumors and remodel the tumor microenvironment. Native cysteines were removed from the parental protein, JO-4, followed by addition of a single cysteine to allow for convenient attachment of various payloads that can be targeted directly to the tumor. Our tumor-targeting protein exhibits high avidity, minimal aggregation, and is easily purified at good yields from E. coli. For proof of concept, we demonstrate effective conjugation to biotin as a model for flexible co-targeting, addition of metal ion chelators as models for imaging and radiotherapy, and linkage of the TLR3 agonist poly(I:C) as a model immune-oncologic agent. This second-generation cancer co-therapeutic protein is optimized for activity and primed for cGMP manufacture in preparation for upcoming clinical studies.

A combination of TLR-4 agonist and saponin adjuvants increases antibody diversity and protective efficacy of a recombinant West Nile Virus antigen.

Members of the Flaviviridae family are the leading causes of mosquito-borne viral disease worldwide. While dengue virus is the most prevalent, the recent Zika virus outbreak in the Americas triggered a WHO public health emergency, and yellow fever and West Nile viruses (WNV) continue to cause regional epidemics. Given the sporadic nature of flaviviral epidemics both temporally and geographically, there is an urgent need for vaccines that can rapidly provide effective immunity. Protection from flaviviral infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. TLR agonist adjuvants represent a promising tool to enhance the protective capacity of flavivirus vaccines through dose and dosage reduction and broadening of antiviral antibody responses. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) using a novel combination adjuvant, which contains a potent TLR-4 agonist and the saponin QS21 in a liposomal formulation (SLA-LSQ). Here, we show that, in combination with WN-80E, optimized SLA-LSQ is capable of inducing long-lasting immune responses in preclinical models that provide sterilizing protection from WNV challenge, reducing viral titers following WNV challenge to undetectable levels in Syrian hamsters. We have investigated potential mechanisms of action by examining the antibody repertoire generated post-immunization. SLA-LSQ induced a more diverse antibody response to WNV recombinant E-protein antigen than less protective adjuvants. Collectively, these studies identify an adjuvant formulation that enhances the protective capacity of recombinant flavivirus vaccines.

Sm-p80-based schistosomiasis vaccine: double-blind preclinical trial in baboons demonstrates comprehensive prophylactic and parasite transmission-blocking efficacy.

Schistosomiasis is of public health importance to an estimated one billion people in 79 countries. A vaccine is urgently needed. Here, we report the results of four independent, double-blind studies of an Sm-p80-based vaccine in baboons. The vaccine exhibited potent prophylactic efficacy against transmission of Schistosoma mansoni infection and was associated with significantly less egg-induced pathology, compared with unvaccinated control animals. Specifically, the vaccine resulted in a 93.45% reduction of pathology-producing female worms and significantly resolved the major clinical manifestations of hepatic/intestinal schistosomiasis by reducing the tissue egg-load by 89.95%. A 35-fold decrease in fecal egg excretion in vaccinated animals, combined with an 81.51% reduction in hatching of eggs into the snail-infective stage (miracidia), demonstrates the parasite transmission-blocking potential of the vaccine. Substantially higher Sm-p80 expression in female worms and Sm-p80-specific antibodies in vaccinated baboons appear to play an important role in vaccine-mediated protection. Preliminary analyses of RNA sequencing revealed distinct molecular signatures of vaccine-induced effects in baboon immune effector cells. This study provides comprehensive evidence for the effectiveness of an Sm-p80-based vaccine for schistosomiasis.

Selection of therapeutic H5N1 monoclonal antibodies following IgVH repertoire analysis in mice.

The rapid rate of influenza virus mutation drives the emergence of new strains that inflict serious seasonal epidemics and less frequent, but more deadly, pandemics. While vaccination provides the best protection against influenza, its utility is often diminished by the unpredictability of new pathogenic strains. Consequently, efforts are underway to identify new antiviral drugs and monoclonal antibodies that can be used to treat recently infected individuals and prevent disease in vulnerable populations. Next Generation Sequencing (NGS) and the analysis of antibody gene repertoires is a valuable tool for Ab discovery. Here, we describe a technology platform for isolating therapeutic monoclonal antibodies (MAbs) by analyzing the IgVH repertoires of mice immunized with recombinant H5N1 hemagglutinin (rH5). As an initial proof of concept, 35 IgVH genes were selected using a CDRH3 search algorithm and co-expressed in a murine IgG2a expression vector with a panel of germline murine kappa genes. Culture supernatants were then screened for antigen binding. Seventeen of the 35 IgVH MAbs (49%) bound rH5VN1203 in preliminary screens and 8 of 9 purified MAbs inhibited 3 heterosubtypic strains of H5N1 virus when assayed by HI. Two of these MAbs demonstrated prophylactic and therapeutic activity in virus-challenged mice. This is the first example in which an NGS discovery platform has been used to isolate anti-influenza MAbs with relevant therapeutic activity.

p28 dynein light chains and ciliary motility in Tetrahymena thermophila.

Dynein light chains are required for the assembly of axonemal dyneins into cilia and flagella. Most organisms express a single p28 dynein light chain and four to nine one-headed inner arm dynein heavy chains. In contrast, Tetrahymena encodes three p28 dynein light chain genes (p28A, p28B, and p28C) and 18 one-headed inner arm dynein heavy chains. In this article it is shown that mutations in p28A and p28B affected both beat frequency and waveform of cilia, while mutations in p28C affected only ciliary beat frequency. A similar set of dynein heavy chains were affected in both p28AKO and p28BKO, but a distinct set of heavy chains was affected in p28CKO. The results suggested that the p28s have non-redundant functions in Tetrahymena and that p28C was associated with a different set of dynein heavy chains than were p28A and p28B.

Development of a schistosomiasis vaccine.

Schistosomiasis is a neglected tropical disease (NTD) of public health importance. Despite decades of implementation of mass praziquantel therapy programs and other control measures, schistosomiasis has not been contained and continues to spread to new geographic areas. A schistosomiasis vaccine could play an important role as part of a multifaceted control approach. With regards to vaccine development, many biological bottlenecks still exist: the lack of reliable surrogates of protection in humans; immune interactions in co-infections with other diseases in endemic areas; the potential risk of IgE responses to antigens in endemic populations; and paucity of appropriate vaccine efficacy studies in nonhuman primate models. Research is also needed on the role of modern adjuvants targeting specific parts of the innate immune system to tailor a potent and protective immune response for lead schistosome vaccine candidates with the long-term aim to achieve curative worm reduction. This review summarizes the current status of schistosomiasis vaccine development.

Label-free electrochemical detection of an Entamoeba histolytica antigen using cell-free yeast-scFv probes.

Inexpensive, simple and quick detection of pathogen antigens in human samples is a key global health objective. Limiting factors include the cost and complexity of diagnostic tests that utilize antibody probes. Herein, we present a method for label-free electrochemical detection of a protein from the enteric pathogen Entamoeba histolytica using cell-free yeast-embedded antibody-like fragments (yeast-scFv) as novel affinity reagents.

Proteomic analysis of the cyst stage of Entamoeba histolytica.

BACKGROUND: The category B agent of bioterrorism, Entamoeba histolytica has a two-stage life cycle: an infective cyst stage, and an invasive trophozoite stage. Due to our inability to effectively induce encystation in vitro, our knowledge about the cyst form remains limited. This also hampers our ability to develop cyst-specific diagnostic tools. AIMS: Three main aims were (i) to identify E. histolytica proteins in cyst samples, (ii) to enrich our knowledge about the cyst stage, and (iii) to identify candidate proteins to develop cyst-specific diagnostic tools. METHODS: Cysts were purified from the stool of infected individuals using Percoll (gradient) purification. A highly sensitive LC-MS/MS mass spectrometer (Orbitrap) was used to identify cyst proteins. RESULTS: A total of 417 non-redundant E. histolytica proteins were identified including 195 proteins that were never detected in trophozoite-derived proteomes or expressed sequence tag (EST) datasets, consistent with cyst specificity. Cyst-wall specific glycoproteins Jacob, Jessie and chitinase were positively identified. Antibodies produced against Jacob identified cysts in fecal specimens and have potential utility as a diagnostic reagent. Several protein kinases, small GTPase signaling molecules, DNA repair proteins, epigenetic regulators, and surface associated proteins were also identified. Proteins we identified are likely to be among the most abundant in excreted cysts, and therefore show promise as diagnostic targets. MAJOR CONCLUSIONS: The proteome data generated here are a first for naturally-occurring E. histolytica cysts, and they provide important insights into the infectious cyst form. Additionally, numerous unique candidate proteins were identified which will aid the development of new diagnostic tools for identification of E. histolytica cysts.