Feasibility and Outcomes of Same-Day Surgery in Primary and Reoperative Laparoscopic Hiatal Hernia Repair.

OBJECTIVE: To assess the feasibility and outcomes of same-day surgery in primary and reoperative laparoscopic hiatal hernia repairs. METHODS: Same-day surgery was planned in elective procedures with ASA II-IV. An Enhanced Recovery After Surgery (ERAS) protocol was implemented to achieve same-day surgery, and opioid-based anesthesia was replaced by opioid-free anesthesia. Outcomes were assessed by length of stay, transition from same-day surgery to observation or inpatient, and postoperative emergency department visits/readmissions. The predictors of same-day surgery were assessed. Values are presented as median (interquartile range). RESULTS: From 04/13/2017 to 09/29/2022, there were 518 laparoscopic hiatal hernia repairs in 491 patients, 428/518 (82.6%) were primary, and 90/518 (17.4%) were reoperative. In the primary group, 314/428 (73.4%) were planned as same-day surgery and 246/314 (78.3%) were performed as same-day surgery. Same-day surgery with opioid-based anesthesia protocol was performed in 77/314 (24.5%) vs. same-day surgery with opioid-free anesthesia protocol in 169/314 (53.8%), p < 0.001, 41/246 (16.7%) same-day surgery primary procedures had emergency department visit post-discharge, and 26/246 (10.6%) were readmitted. In the reoperative group, 51/90 (56.7%) were planned as same-day surgery, and 27/51 (52.9%) were performed as same-day surgery. Same-day surgery with opioid-based anesthesia protocol was performed in 2/51 (3.9%) vs. same-day surgery with opioid-free anesthesia protocol in 25/51 (49.0%), p < 0.001, 3/27 (11.1%) same-day surgery reoperative procedures had emergency department visit post-discharge, and 3/27 (11.1%) were readmitted. Opioid-free anesthesia protocol was the positive predictor of same-day surgery compared to opioid-based anesthesia protocol (OR 7.44 [95% CI: 2.94, 18.83]), p < 0.001. Negative predictors were ASA III compared to II (OR 0.52 [95% CI: 0.28, 0.94]), p = 0.031, and duration of operation (OR 0.98 [0.97, 0.99]) p < 0.001. CONCLUSION: Laparoscopic hiatal hernia repair can be performed as same-day surgery in the majority of primary and reoperative procedures with good outcomes and low postoperative emergency department visits and readmissions. The odds of same-day surgery are higher with opioid-free anesthesia, lower ASA, and shorter operative time.

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni.

The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.

Opioid-Free Anesthesia and Same-Day Surgery Laparoscopic Hiatal Hernia Repair.

BACKGROUND: Laparoscopic hiatal hernia repair is commonly performed with a 1 to 2 night hospitalization. Our aim was to compare the feasibility and short-term outcomes of same-day surgery (SDS) laparoscopic hiatal hernia repair with an opioid-based anesthesia protocol (OBAP) vs an opioid-free anesthesia protocol (OFAP). STUDY DESIGN: Outcomes and pharmacy costs of repairs with OBAP were compared with OFAP. Values were expressed as median (interquartile range) and costs as means. RESULTS: There were 244 primary laparoscopic repairs. OBAP was used in 191 of 244 (78.3%) vs OFAP in 53 of 244 (21.7%). The length of stay was 1 day (0 to 2) vs 0 days (0 to 1), p = 0.006. There was no difference between the percentage of patients requiring analgesics and dosage between the 2 groups. SDS was planned in 157 and performed in 74 of 122 (60.7%) vs 33 of 35 (94.3%), p < 0.001. The age was 56 years (45 to 63) vs 60 years (56 to 68), p = 0.025. There were more type I hiatal hernia in SDS-OBAP and more type III and IV in SDS-OFAP, p = 0.031. American Society of Anesthesiologists Physical Status was II (II-III) vs III (II-III), p = 0.045. SDS was not performed in 50 of 157 (31.8%), 48 of 122 (39.3%) vs 2 of 35 (5.7%), p < 0.001. Out of 157 planned SDS, nausea/retching were causes of transition in 19 of 122 (15.6%) vs 0 of 35 (0%), p = 0.020. Multivariable logistic regression showed the odds of SDS were 8.21 times (95% CI 3.10 to 21.71; p < 0.001) greater in OFAP compared with OBAP, adjusting for sex, age, body mass index, American Society of Anesthesiologists Physical Status, type of hiatal hernia, type of procedure, and duration of the operation. Patients with opioid medication after SDS discharge were 74 of 74 (100%) vs 22 of 33 (66.7%), p < 0.001. CONCLUSIONS: Opioid-free anesthesia increases the feasibility of SDS hiatal hernia repair with less perioperative nausea and comparable pain control and pharmacy cost.

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni.

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

Schistosome AMPK Is Required for Larval Viability and Regulates Glycogen Metabolism in Adult Parasites.

On entering the mammalian host, schistosomes transition from a freshwater environment where resources are scarce, to an environment where there is an unlimited supply of glucose, their preferred energy substrate. Adult schistosome glycolytic activity consumes almost five times the parasite's dry weight in glucose per day to meet the parasite's energy demands, and the schistosome glycolytic enzymes and mechanisms for glucose uptake that sustain this metabolic activity have previously been identified. However, little is known of the parasite processes that regulate schistosome glucose metabolism. We previously described the Schistosoma mansoni ortholog of 5' AMP-Activated Protein Kinase (AMPK), which is a central regulator of energy metabolism in eukaryotes, and characterized the developmental regulation of its expression and activity in S. mansoni. Here we sought to explore the function of AMPK in schistosomes and test whether it regulates parasite glycolysis. Adult schistosomes mounted a compensatory response to chemical inhibition of AMPK α, resulting in increased AMPK α protein abundance and activity. RNAi inhibition of AMPK α expression, however, suggests that AMPK α is not required for adult schistosome viability in vitro. Larval schistosomula, on the other hand, are sensitive to chemical AMPK α inhibition, and this correlates with inactivity of the AMPK α gene in this life cycle stage that precludes a compensatory response to AMPK inhibition. While our data indicate that AMPK is not essential in adult schistosomes, our results suggest that AMPK regulates adult worm glycogen stores, influencing both glycogen utilization and synthesis. AMPK may therefore play a role in the ability of adult schistosomes to survive in vivo stressors such as transient glucose deprivation and oxidative stress. These findings suggest that AMPK warrants further investigation as a potential drug target, especially for interventions aimed at preventing establishment of a schistosome infection.

Laparoscopic hiatal hernia repair as same day surgery: Feasibility, short-term outcomes and costs.

INTRODUCTION: Laparoscopic hiatal hernia repair is commonly performed with 1 night hospitalization. The aim was to assess repairs as same-day-surgery (SDS). METHODS: Costs/short-term outcomes of SDS were compared to hospital-stay < 24-h: observation (OBS) and hospital-stay ≥ 24-h: inpatient (INP). Outcomes were assessed by postoperative 30-day ER visits/readmissions. RESULTS: There were 262 procedures, excluding 50 reoperative repairs, 212 procedures were included: There were 66 SDS, 65 OBS and 81 INP. SDS vs. OBS: OBS were older, had higher ASA, less type I and more type III and IV hernias. Costs were significantly less in the SDS group with no difference in post-operative ER visits/post-discharge readmissions. SDS vs. INP: INP were older, had higher ASA, less type I and more type III and IV hernias. Costs were significantly less in the SDS group with no difference in post-operative ER visits/post-discharge readmissions. CONCLUSION: Laparoscopic hiatal hernia repair can be performed as SDS in majority of elective repairs with good short-term outcomes and reduced cost.

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor.

BACKGROUND: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. METHODS: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. RESULTS: Gene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01). CONCLUSIONS: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

Laparoscopic Heller myotomy and Dor fundoplication in the same day surgery setting with a trained team and an enhanced recovery protocol.

BACKGROUND: The length of stay after Heller myotomy is 1-5 days. The aim was to report feasibility of the procedure as same day surgery (SDS). METHODS: Three steps of Enhanced Recovery After Surgery protocol: preoperatively, clear liquid diet for 24 hours, in preoperative area: antiemetics as dermal patch/IV form, 2: Intraoperatively, intubation in semi upright position, IV analgesics and antiemetics. 3: Postoperatively, clear liquid diet and discharge instructions. Patients were followed using a phone questionnaire. Values are median (interquartile range). RESULTS: Fifty-seven patients, 32 M (56%)/25F (44%), age 48 (35-59). First 45 were inpatient with LOS of 1 day. Last 12 were planned as same day surgery, 1/12 was discharged on POD#2, 11/12 (92%) were performed as same day surgery. The duration of operation: 139.5 min (114-163) inpatient: vs 123 (107-139) same day surgery, P < .01. Questionnaires were obtained in 78% inpatient at 40 months (25.6-67) vs 82% same day surgery at 8 (4-12). All were satisfied with the operation with no difference between the 2 groups. CONCLUSION: Heller myotomy can be planned as same day surgery and performed successfully in majority of patients with a trained team and an Enhanced Recovery After Surgery protocol focused on prevention of nausea, and pain control in perioperative period.

Patient perspectives of an individualized diabetes care management plan.

PURPOSE: This cross sectional study examines patients' knowledge, attitudes and beliefs about a diabetic care management plan (DCMP) that was developed to provide patient education on diabetes guidelines and display individual diabetic core measures. Secondary objectives included a comparison of diabetic core measures [hemoglobin A1C (HbA1C), systolic and diastolic blood pressure (SBP, DBP), low-density lipoprotein (LDL) and urine microalbumin (Um)] before and after DCMP implementation. We hypothesize this tool will contribute to patients' awareness of current disease status, diabetes knowledge and diabetic core value improvement over time. METHODS: A consecutive sample of 102 adult patients with diabetes mellitus type 2 in a primary care setting participated. Patients' perspectives on the care plan and knowledge about diabetes was collected via survey after care plan implementation. A comparison of selected diabetic core measures was conducted at baseline and post-DCMP. Descriptive statistics summarized survey response and diabetic core measures. A repeated measures ANOVA was used to assess change in diabetic core measures over time. RESULTS: Participants understood the DCMP (96%), found it important because it explained their laboratory results and medications (89%) and believed it would help them to have better diabetic control (99%). There was a significant interaction between time and being at goal pre-DCMP for HbA1c, SBP and LDL. Patients not at goal pre-DCMP for the above measures decreased significantly over time (P = <0.01 for HbA1c, SBP and LDL). Participants at goal for all diabetic core measures increased pre- to post-DCMP from 13% to 20% (P = 0.28). CONCLUSION: Patients perceived the diabetic care management plan favorably and their diabetic core measurements improved over time. This simple and reproducible self-management intervention can enhance self-management in a patient population with diabetes mellitus type 2.

Effect of Food Waste Co-Digestion on Digestion, Dewatering, and Cake Quality.

The objective of this study was to evaluate the effect of food waste addition on anaerobic digestion performance as well as downstream parameters including dewatering, cake quality, and filtrate quality. Laboratory-scale digesters were fed processed food waste at rates of 25%, 45%, and 65% increased chemical oxygen demand (COD) loading rates compared to a control fed only primary and secondary solids. The specific methane yield increased from 370 L CH4/kg VSadded for the control to 410, 440, and 470 L CH4/kg VSadded for the 25, 45, and 65% food waste addition, respectively. The cake solids after dewatering were all higher for the food waste digesters compared to the control, with the highest cake solids being measured for the 45% food-waste loading. Compared to the control digester, the biosolids odorant concentration increased for the lowest dose of food waste. Odorant concentrations were below detection for the highest food waste loading.

The NIH-NIAID Schistosomiasis Resource Center at the Biomedical Research Institute: Molecular Redux.

Schistosomiasis remains a health burden in many parts of the world. The complex life cycle of Schistosoma parasites and the economic and societal conditions present in endemic areas make the prospect of eradication unlikely in the foreseeable future. Continued and vigorous research efforts must therefore be directed at this disease, particularly since only a single World Health Organization (WHO)-approved drug is available for treatment. The National Institutes of Health (NIH)-National Institute of Allergy and Infectious Diseases (NIAID) Schistosomiasis Resource Center (SRC) at the Biomedical Research Institute provides investigators with the critical raw materials needed to carry out this important research. The SRC makes available, free of charge (including international shipping costs), not only infected host organisms but also a wide array of molecular reagents derived from all life stages of each of the three main human schistosome parasites. As the field of schistosomiasis research rapidly advances, it is likely to become increasingly reliant on omics, transgenics, epigenetics, and microbiome-related research approaches. The SRC has and will continue to monitor and contribute to advances in the field in order to support these research efforts with an expanding array of molecular reagents. In addition to providing investigators with source materials, the SRC has expanded its educational mission by offering a molecular techniques training course and has recently organized an international schistosomiasis-focused meeting. This review provides an overview of the materials and services that are available at the SRC for schistosomiasis researchers, with a focus on updates that have occurred since the original overview in 2008.

Cardiovascular and cerebrovascular responses to progressive central hypovolemia in young smokers: a preliminary study.

The purpose of this study was to describe cardiovascular and cerebrovascular responses of smokers and nonsmokers to progressive central hypovolemia. Twenty subjects participated (equal male and female). We recorded the electrocardiogram, beat-to-beat arterial pressure (Finometer), cerebral blood velocity of the middle cerebral artery (transcranial Doppler), and end-tidal CO2. Lower body negative pressure (LBNP) was applied at 3 mm Hg · min(-1) for 20 minutes to an ending pressure of -60 mm Hg, and data were averaged in 2-minute bins. Arterial pressures were similar between groups at baseline, but heart rates tended to be higher, and stroke volumes and cerebral velocities tended to be lower in smokers at baseline and during LBNP (all p ≥ 0.17). Heart rates increased, and arterial pressures, stroke volumes, and cerebral velocities decreased during LBNP (all p ≤ 0.05), but responses were not different between smokers and nonsmokers. During the final stage of LBNP, systolic pressures and mean middle cerebral artery velocities were substantially lower in smokers than nonsmokers: these preliminary data may suggest clinical relevance of smoking status, but the magnitude of differences between groups were not distinguishable statistically. We therefore conclude that smokers and nonsmokers respond similarly to progressive central hypovolemia.

Schistosomes and snails: a molecular encounter.

Biomphalaria glabrata snails play an integral role in the transmission of Schistosoma mansoni, the causative agent for human schistosomiasis in the Western hemisphere. For the past two decades, tremendous advances have been made in research aimed at elucidating the molecular basis of the snail/parasite interaction. The growing concern that there is no vaccine to prevent schistosomiasis and only one effective drug in existence provides the impetus to develop new control strategies based on eliminating schistosomes at the snail-stage of the life cycle. To elucidate why a given snail is not always compatible to each and every schistosome it encounters, B. glabrata that are either resistant or susceptible to a given strain of S. mansoni have been employed to track molecular mechanisms governing the snail/schistosome relationship. With such snails, genetic markers for resistance and susceptibility were identified. Additionally, differential gene expression studies have led to the identification of genes that underlie these phenotypes. Lately, the role of schistosomes in mediating non-random relocation of gene loci has been identified for the first time, making B. glabrata a model organism where chromatin regulation by changes in nuclear architecture, known as spatial epigenetics, orchestrated by a major human parasite can now be investigated. This review will highlight the progress that has been made in using molecular approaches to describe snail/schistosome compatibility issues. Uncovering the signaling networks triggered by schistosomes that provide the impulse to turn genes on and off in the snail host, thereby controlling the outcome of infection, could also yield new insights into anti-parasite mechanism(s) that operate in the human host as well.

Identification and characterisation of functional expressed sequence tags-derived simple sequence repeat (eSSR) markers for genetic linkage mapping of Schistosoma mansoni juvenile resistance and susceptibility loci in Biomphalaria glabrata.

Biomphalaria glabrata susceptibility to Schistosoma mansoni has a strong genetic component, offering the possibility for investigating host-parasite interactions at the molecular level, perhaps leading to novel control approaches. The identification, mapping and molecular characterisation of genes that influence the outcome of parasitic infection in the intermediate snail host is, therefore, seen as fundamental to the control of schistosomiasis. To better understand the evolutionary processes driving disease resistance/susceptibility phenotypes, we previously identified polymorphic random amplification of polymorphic DNA and genomic simple sequence repeats from B. glabrata. In the present study we identified and characterised polymorphic expressed simple sequence repeats markers (Bg-eSSR) from existing B. glabrata expressed sequence tags. Using these markers, and with previously identified genomic simple sequence repeats, genetic linkage mapping for parasite refractory and susceptibility phenotypes, the first known for B. glabrata, was initiated. Data mining of 54,309 expressed sequence tag, produced 660 expressed simple sequence repeats of which dinucleotide motifs (TA)n were the most common (37.88%), followed by trinucleotide (29.55%), mononucleotide (18.64%) and tetranucleotide (10.15%). Penta- and hexanucleotide motifs represented <3% of the Bg-eSSRs identified. While the majority (71%) of Bg-eSSRs were monomorphic between resistant and susceptible snails, several were, however, useful for the construction of a genetic linkage map based on their inheritance in segregating F2 progeny snails derived from crossing juvenile BS-90 and NMRI snails. Polymorphic Bg-eSSRs assorted into six linkage groups at a logarithm of odds score of 3. Interestingly, the heritability of four markers (Prim1_910, Prim1_771, Prim6_1024 and Prim7_823) with juvenile snail resistance were, by t-test, significant (P<0.05) while an allelic marker, Prim24_524, showed linkage with the juvenile snail susceptibility phenotype. On the basis of our results it is possible that the gene(s) controlling juvenile resistance and susceptibility to S. mansoni infection in B. glabrata are not only on the same linkage group but lie within a short distance (42cM) of each other.

Polyethyleneimine (PEI) mediated siRNA gene silencing in the Schistosoma mansoni snail host, Biomphalaria glabrata.

An in vivo, non-invasive technique for gene silencing by RNA interference (RNAi) in the snail, Biomphalaria glabrata, has been developed using cationic polymer polyethyleneimine (PEI) mediated delivery of long double-stranded (ds) and small interfering (si) RNA. Cellular delivery was evaluated and optimized by using a 'mock' fluorescent siRNA. Subsequently, we used the method to suppress expression of Cathepsin B (CathB) with either the corresponding siRNA or dsRNA of this transcript. In addition, the knockdown of peroxiredoxin (Prx) at both RNA and protein levels was achieved with the PEI-mediated soaking method. B. glabrata is an important snail host for the transmission of the parasitic digenean platyhelminth, Schistosoma mansoni that causes schistosomiasis in the neotropics. Progress is being made to realize the genome sequence of the snail and to uncover gene expression profiles and cellular pathways that enable the snail to either prevent or sustain an infection. Using PEI complexes, a convenient soaking method has been developed, enabling functional gene knockdown studies with either dsRNA or siRNA. The protocol developed offers a first whole organism method for host-parasite gene function studies needed to identify key mechanisms required for parasite development in the snail host, which ultimately are needed as points for disrupting this parasite mediated disease.

Non-random organization of the Biomphalaria glabrata genome in interphase Bge cells and the spatial repositioning of activated genes in cells co-cultured with Schistosoma mansoni.

Biomphalaria glabrata is a major intermediate host for the parasitic trematode Schistosoma mansoni, a causative agent of human schistosomiasis. To decipher the molecular basis of this host-parasite interaction, the Bge embryonic cell line provides a unique in vitro model system to assess whether interactions between the snail and parasite affect the cell and genome biology in either organism. The organization of the B. glabrata genome in Bge cells was studied using image analysis through positioning territories of differently sized chromosomes within cell nuclei. The snail chromosome territories are similar in morphology as well as in non-random radial positioning as those found in other derived protostome and deuterostome organisms. Specific monitoring of four gene loci, piwi, BgPrx, actin and ferritin, revealed non-random radial positioning of the genome. This indicates that specific parts of the snail genome reside in reproducible nuclear addresses. To determine whether exposure to parasite is reflected in genome organization, the interphase spatial positioning of genes was assessed after co-culturing Bge cells with either normal or irradiation attenuated miracidia for 30 min to 24 h. The loci of actin and ferritin, genes that are up-regulated in the snail when subjected to infection, were visualized by fluorescence in situ hybridisation (FISH) and their radial nuclear positions i.e. their position in the interphase nucleus with respect to the nuclear edge/envelope, mapped. Interestingly, large scale gene repositioning correlated to temporal kinetics of gene expression levels in Bge cells co-cultured with normal miracidia while irradiated parasites failed to elicit similar gene expression or gene loci repositioning as demonstrated using the ferritin gene. This indicates that normal but not attenuated schistosomes provide stimuli that evoke host responses that are reflected in the host's nuclear architecture. We believe that this is not only the first time that gene-repositioning studies have been attempted in a mollusc but also demonstrates a parasite influencing the interphase genome organization of its host.

FISH on chromosomes derived from the snail model organism Biomphalaria glabrata.

The application of fluorescence in situ hybridization (FISH) for the mapping of single copy genes onto homologous chromosome has been integral to vast number genome sequencing projects, such as that of mouse and human. The chromosomes of these organisms are well-studied and are the staple resource of most of the early studies conducted in cytogenetics. However, there are now protocols for analyzing FISH probes in a number of different organisms on both metaphase and interphase chromosomes.Here, we describe the methodologies for the chromosomal mapping of nonrepetitive (single-copy) genes of the snail Biomphalaria glabrata onto metaphase chromosomes derived from the only molluscan cell-line in existence. The technique described in this chapter was developed for the B. glabrata genome sequencing project through troubleshooting experimental procedures established for other organisms so that both the optimum resolution of metaphase chromosome and the effective hybridization of genes were achieved.

Identification of immediate response genes dominantly expressed in juvenile resistant and susceptible Biomphalaria glabrata snails upon exposure to Schistosoma mansoni.

Resistance or susceptibility of the snail host Biomphalaria glabrata to Schistosoma mansoni is determined by the genetics of both the snail and parasite. Although Mendelian genetics governs adult resistance to infection, juvenile resistance and susceptibility are complex traits. In this study, suppression subtractive hybridization was used to construct forward and reverse cDNA libraries to identify genes involved in the immediate response of juvenile resistant (BS-90), non-susceptible (LAC2) snails, and susceptible (NMRI) snails after early exposure to S. mansoni. Expressed Sequence Tags (ESTs) were generated from the repertoire of enriched transcripts. In resistant snails, several ESTs corresponded to transcripts involved in immune regulation/defense response. While no defense related transcripts were found among juvenile susceptible snail ESTs, we detected transcripts involved in negative regulation of biological process/morphogenesis/proliferation. Differential gene expression and temporal regulation of representative transcripts were compared among snails pre- and post-exposure to either normal or attenuated miracidia using quantitative real time RT-PCR. Results showed that several transcripts, such as fibrinolytic C terminal domain, cytidine deaminase, macrophage expressed gene 1, protein kinase C receptor, anti-microbial peptide; theromacin and Fas remained up-regulated regardless of whether or not snails were exposed to normal or attenuated miracidia. While ESTs related to C-type lectin and low-density lipoprotein receptor were induced only by exposure to normal miracidia. By comparing changes in gene expression between resistant and susceptible juvenile snails responding either to normal or attenuated parasites, we can conclude that the transcription of genes associated with the intra-dermal penetration process of the snail host by invading miracidia may need to be taken into account when assessing differential gene expression between resistant and susceptible strains of B.glabrata in relation to S. mansoni exposure.

Schistosoma mansoni infection of juvenile Biomphalaria glabrata induces a differential stress response between resistant and susceptible snails.

Schistosomes develop successfully in susceptible snails but are encapsulated and killed in resistant ones. Mechanism(s) shaping these outcomes involves the parasites ability to evade the snail's defenses. RNA analysis from resistant (BS-90), non-susceptible (LAC2) and susceptible (NMRI) juvenile Biomphalaria glabrata to Schistosoma mansoni revealed that stress-related genes, heat shock protein 70 (Hsp 70) and reverse transcriptase (RT), were dramatically co-induced early in susceptible snails, but not in resistant/non-susceptible ones. These transcripts were, however, down regulated upon exposure to irradiated parasites although penetration behavior of irradiated vs. normal parasites were the same, indicating that Hsp 70 and RT regulation was elicited by infection and not injury. Understanding molecular events involved in stress response transcriptional regulation of Hsp 70 in juvenile snails could pave a way towards the identification of genes involved in schistosome/snail interactions.