This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids, and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT) domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR's cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs) in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.Ca In 1993, Dr. Edward M. Brown cloned CaSR, which is primarily responsible for this type of regulation, from bovine parathyroid gland , CaSR has also been reported to be present in diverse other, non-homeostatic tissues   and γ-aminobutyric acid (GABA)B receptors. As shown in Figure 2+]o triggers multiple CaSR-regulated intracellular signaling pathways, including Gq∕11 signaling, Gi∕osignaling, Gs signaling, extracellular signal-regulated kinases 1 and 2 (ERK1∕2) signaling, and intracellular calcium mobilization.In 1883, Sydney Ringer serendipitously discovered Ca2+, L-Phe and polypeptides, allowing ligands to modulate CaSR cooperatively, but is also essential for the dimerization of the receptor  N-terminal signal peptide, a ~600 AA extracellular domain (ECD), a cysteine rich domain (CRD), a ~250 AA 7-transmembrane (7TM) domain, and the ~216 AA intracellular domain (ICD)   and limitations in obtaining purified membrane proteins with native conformations , which then causes the signal to be relayed to the 7TM domain. As cGPCRs are only functional as dimers, the dimerization of CaSR is likely to be driven by the hydrophobic interactions between the monomers along the dimer interface. CaSR and mGluR protomers are also covalently linked by several disulfide bridges between the two monomers. A ligand induced rearrangement of the dimeric VFT brings the two monomers closer together by pulling the lower lobes inwards upon the change toward the closed state. Similar to mGluR, the ligand was proposed to interact with both lobes of the VFT to stabilize the closed form by providing additional points of contact between the lobes  have been provided by different independent groups  is located within the hinge region between the two lobes of the ECD , and Site 5, which is formed by contiguous Ca2+ binding residues , were verified using a grafting approach  fluorescence, intrinsic tryptophan fluorescence spectra and nuclear magnetic resonance (NMR)  were purified from HEK293S and its mutant cell line P3 (IP3), which binds to the IP3 receptor on the ER, releasing stored Ca2+. On the other hand, L-Phe in the presence of Ca2+ induces coupling to heterotrimeric GTP binding protein G12∕13, leading to the activation of RhoA via filamin A.The extracellular calcium induced activation of CaSR is potentiated by L-amino acids to maintain the whole body Ca2+]i mobilization using a fluorescence-based cell population assay. Meanwhile, studies from Mun et al. showed the binding site for amino acids was within the VFT domain of CaSR by utilizing CaSR-mGluR chimeric receptor constructs. The receptor lacking the ECD exhibited impaired response to L-amino acids  which is different than those produced by Ca2+ with L-amino acid  (2.2/min)   and several FHH mutations  are within Ca2+ binding site 1. The FHH mutation D215G is located in Ca2+ binding site 2 while two ADH mutations E228Q and E228K are in Site 3. The Ca2+ binding Site 4 embraces residues involved in a carcinoma-associated mutation E350V and an ADH mutation E354A. Mutations on these key binding residues leads to a decrease of the EC50 of Ca2+ in ADH in order to produce PTH, and an increase of EC50 in FHH or NSHPT. This can also lead to an alteration of binding cooperativity between the other Ca2+ binding sites, allowing them to bind more or less effectively as indicated by changes in the Hill number. Several studies have investigated the effect of FHH Ca2+ binding site 1 mutations, such as Y218S  accumulation, but does not change CaSR cell expression levels as compared to the wild-type (WT)  and E191K (ADH)  activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca2+]o change. Further analysis of the dynamics of the VFT domain mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. WT CaSR was modulated by L-Phe in a heterotropic positively cooperative way, achieving an EC50 for Ca2+ induced intracellular responses similar to those of the two activating mutations. The response of the inactivating P221Q mutant to Ca2+ was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and within intact cells indicate that residue Leu173, which is adjacent to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, P221 and L173 are important for the positive homo—and heterotropic cooperative regulation elicited by agonist binding.The second type of disease mutations, type II, disrupts molecular connectivity embedded into the hinge region of the ECD Figures , 5. Muta50 for extracellular Ca2+ elicited intracellular response of the CaSR are also determined by the receptor expression level at the plasma membrane , which may lead to the retention of CaSR in the ER  contributed to the hallmarks of CaSR signaling, including the high degree of cooperativity and the lack of functional desensitization. Additionally, they have shown that the life cycle of the CaSR controls the cellular abundance of CaSR, suggesting an intimate link between trafficking and signaling  has been shown to be effective for the treatment of osteoporosis  on a pH dependent manner where slightly lower extracellular pH of 7.2 inhibits the binding of the ligand to CaSR, and a higher pH of 7.6 favors the binding process , to induce intracellular Ca2+ mobilization, were in the range of sub-micromolar   to be positive allosteric modulators of CaSR. They also found that the double mutant T145A/S170T had an impaired response to these peptides, indicating a potential peptide binding site at the extracellular domain of CaSR , participating in various functions, such as Ca2+ signaling could be modulated through the formation of CaSR hetero-dimers with other GPCRs or higher order oligomers with non GPCR chaperones in tissues other than parathyroid. Gama et al. showed co-localization of the CaSR and mGluR1α in hippocampal and cerebellar neurons by immunoprecipitation of CaSR from bovine brain. The CaSR became sensitive to glutamate triggering internalization and exhibited altered trafficking via reported hetero-dimerization with GABAB receptors  in hippocampal neurons , and funds from the Georgia Research Alliance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RUNX1 is a recurrently mutated gene in sporadic myelodysplastic syndrome and leukemia. Inherited mutations in RUNX1 cause familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). In sporadic AML, mutations in RUNX1 are usually secondary events, whereas in FPD/AML they are initiating events. Here we will describe mutations in RUNX1 in sporadic AML and in FPD/AML, discuss the mechanisms by which inherited mutations in RUNX1 could elevate the risk of AML in FPD/AML individuals, and speculate on why mutations in RUNX1 are rarely, if ever, the first event in sporadic AML. DNMT3A, ASXL1, IDH2, and TET2 , signaling molecules , splicing factors , and proteins with other functions   that promote their clonal expansion and impair downstream differentiation. The earliest initiating step in leukemia is a mutation that generates a hematopoietic stem cell (HSC) that is, in some poorly understood way, primed for leukemic transformation by secondary mutations. The mutations in HSCs that initiate sporadic leukemia in adults generally occur in genes encoding epigenetic regulatory proteins such as RUNX1 mutations; however, the lifetime risk approaches nearly 100% for germ line CEBPA mutations  are also frequently found in sporadic acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) , acute lymphocytic leukemia (ALL), atypical chronic myeloid leukemia (aCML), and secondary AML  in de novo AML and t in acute lymphocytic leukemia (B-ALL). Both of these translocations are initiating events in sporadic leukemia and can be acquired in utero as evidenced by their presence in Guthrie card samples from newborns diagnosed in childhood with AML or B-ALL  to reflect the possible worse prognosis as compared to other AML types  is a clonal hematopoietic disorder characterized by ineffective hematopoiesis and is a harbinger of AML in approximately 25–30% of patients   were also rare or undetected.The paucity of initiating RUNX1 mutations are more common in clonal cytopenia of undetermined significance (CCUS)  , inv(16)/t(16), and t, presumably because the mutations are either functionally redundant or antagonistic. Other common MDS or AML mutations  were neither enriched nor inversely correlated with RUNX1 mutations.The ability to monitor HSC expansion by changes in variant allele frequencies in FPD/AML individuals by next generation sequencing may offer the opportunity to intervene at the pre-leukemic stage, prior to the appearance of overt MDS or leukemia. Knowing when to intervene and treat a healthy individual at an early stage of disease, however, requires knowing which secondary mutations are likely to lead to disease progression. In sporadic leukemia, it is assumed that the most potent cooperating mutations are those that most frequently co-occur due to selection. For CDC25C in 4 patients, concluding that somatic mutations in CDC25C (which are rarely observed in sporadic AML) were the most common genetic event in AML arising from FPD/AML  from the United States analyzed somatic mutations in 7 FPD/AML patients with MDS or AML and found no mutations in either CDC25C or in the second RUNX1 allele, but instead, identified mutations in a collection of other genes including BCOR, PHF6, DNMT3A, TET2, CREBBP, U2AF1, NUP214, SMC3, and PDS5B , base excision (BER), nucleotide excision (NER), double-strand break repair , and interstrand DNA crosslink repair by the Fanconi Anemia (FA) / BReast CAncer susceptibility (BRCA) pathway  protein. Several studies showed that expression of RUNX1-RUNX1T1 in primary HSPCs or cell lines resulted in down regulation of a number of genes involved in BER, including in vitro gradually acquired more γ-H2AX foci than normal cells over time, indicating that they more rapidly accumulated unrepaired double-strand breaks  polymerase (PARP) inhibitors  to promote leukemogenesis was greatly enhanced by transplantation into old recipient mice, whereas when transplanted into young hosts, leukemogenesis was largely suppressed  linked et al. showed that RUNX1 interacted with the IκB kinase (IKK) complex in the cytoplasm, and deletion of Runx1 activated NF-κB signaling  and committed myeloid progenitors in the bone marrow  in the bone marrow of both young and aged mice , Runx1+/−, and wild type HSCs performed equivalently well in serial transplantation experiments, in that by the fourth round of transplantation, HSCs of all three genotypes were essentially exhausted, and hence had similar self-renewal potential  and normal bone marrow cells followed by transplantation into mice resulted in a greater selective expansion of Runx1 deficient HSPCs (lineage negative Sca1+ Kit+ cells) and myeloid lineage cells in the bone marrow compared to irradiated wild type or non-irradiated Runx1 deficient cells (Cai et al., Runx1 deficient HSPCs to endogenous and genotoxic stress (Cai et al., Tp53 mutation, consistent with this interpretation (Marusyk et al., Runx1 deficient HSPCs were less apoptotic, grew more slowly than normal HSCs, had a lower rate of protein translation, and had markedly decreased ribosome biogenesis. Consistent with decreased ribosome biogenesis, Runx1 deficient HSPCs were smaller than normal HSPCs, and preliminary data suggested that this small cell phenotype was shared with HSPCs from FPD/AML individuals (Cai et al., RUNX1 mutations confer a relatively poor prognosis in AML. It may also explain the observation that RUNX1 mutations occurred at a higher frequency in MDS patients previously exposed to low-level radiation as compared to all MDS patients (Harada et al., An interesting and potentially clinically consequential feature of RUNX1 mutant HSPCs is that they exhibit a greater selective advantage over normal HSPCs following exposure to radiation. Irradiation of a mixture of RUNX1 mutations cause leukemia predisposition because every HSC in an FPD/AML individual is already one mutation along the path to AML. However, it is likely that inherited RUNX1 mutations play a more active role in promoting leukemia progression. The dysregulation of DNA repair and decreased apoptosis of RUNX1 mutant HSCs along with an increased inflammatory microenvironment may contribute to the markedly increased incidence of MDS, AML, and T-ALL in FPD/AML individuals. We envision a model whereby RUNX1 mutations increase the rate at which secondary mutations are acquired; moreover, increased inflammatory signals delivered by RUNX1 mutant myeloid lineage cells or other cells in the RUNX1 mutant bone marrow microenvironment provide a selective pressure that confers a competitive advantage to FPD/AML HSCs that have acquired secondary mutations (Figure RUNX1 mutation does not have a competitive advantage over normal HSCs and hence does not expand and initiate leukemia.It was originally thought that inherited DNMT3A mutations have been reported in two asymptomatic FPD/AML individuals (Churpek et al., DNMT3A mutation, which in a normal individual only modestly increases the risk of leukemia, confer a much greater risk in the context of an inherited RUNX1 mutation? More data and longitudinal studies correlating the stepwise accumulation of mutations in individuals with FPD/AML with clinical outcome are needed to predict which mutations most likely portend a rapid progression to MDS or AML. Mechanistic studies in animal and cell models will also be necessary to understand what is driving leukemia progression in FPD/AML and to identify strategies that can avert, delay, or reverse this progression.The initiation and evolution of leukemia in FPD/AML individuals can be carefully followed by next generation sequencing. Individuals diagnosed with FPD/AML could be monitored on a regular basis for evidence of clonal HSC expansion driven by acquired secondary mutations and treated before they progress to frank AML. However, we still do not know which secondary mutations are the most deleterious and whether they are the same as or different than the co-occurring mutations in sporadic AML. For example, All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acute myelogenous leukemia (AML) accounts for 1.2% of all cancer deaths. Relapse is the major cause of treatment failure in acute myeloid leukemia (AML) patients. AML rarely presents as ocular manifestation in relapse or at presentation. The M4 subtype of AML is most commonly presented with extramedullary involvement. In this report, we presented a young female with AML who was diagnosed and treated for AML about 40 months ago. She did not transplant because she did not have a full-match donor. About 4 months ago, she visited with a red eye and conjunctival infiltration. She was referred to an ophthalmologist for a biopsy, and the pathology report showed the relapse of AML which was treated with systemic chemotherapy. Red eyes with subconjunctival nodules in patients with a history of previous AML should raise the suspicion for recurrent disease that warrants urgent biopsy and systemic treatment. Eye involvement with leukemia is usually responsive to systemic chemotherapy. Long-term survival rates in acute myeloid leukemia have been markedly improved by intensification of first-line treatment and better supportive care with overall survival rates increasing from 40 to 60% during the past two decades. However, relapse remains a major obstacle for improving prognosis further, and also, we can see unusual relapse. AML has only rarely been reported as causing ocular complications. Most ocular infiltrates from AML include chorioretinal or optic nerve lesions and are found incidentally in patients with advanced disease , 2. BaseAn 18-year-old woman with a history of documented AML was admitted in Imam-Reza Hospital in Mashhad (Iran) in December 2014. The bone marrow specimen consisted of more than 70% myelomonoblast (M4), which was positive for MPO, CD13, CD14, and CD33 in flow cytometry study, and also she had normal cytogenetics. She underwent induction chemotherapy with 3 + 7 regimen daunorubicin + ara C and achieved complete remission, after that she took intensification chemotherapy for 3 cycles. She was candidated for hematopoietic stem cell transplantation, but she was a single child and did not have a full-match donor. Therefore, we searched for unrelated full-match donor, but we could not find one and she underwent 6-thioguanine as maintenance.She presented with complaints of painful and bilateral red eyes after three and half years . She hadHer leukocyte count and differentiation, Hb, and platelet count were normal. Peripheral blood smear and bone marrow examination were done, and she was in complete remission. Biopsy was taken from her conjunctival lesion, and infiltration of blast cells was reported . SubsequIn patients with acute leukemia, it has been necessary to take a closer look at the site of extramedullary leukemic infiltration because of local morbidity and also because they might act as a reservoir for proliferation of leukemic cells and eventual systemic relapse. Ophthalmic findings are rare findings as an initial manifestation of new or relapsed disease . Eyes arThe M4 myeloid leukemia subtype consists of about 20% of all AML cases, and it is associated with extramedullary infiltration most frequently in gingiva, skin, and central nervous system in comparison to other subtypes . Ocular In two prospective studies of 116 patients diagnosed with AML, none of them were found to have leukemic infiltration in their eyes. Ocular manifestations of leukemia may present as direct infiltration of the uvea, conjunctiva, and optic nerve or as secondary to the hematologic changes of leukemia such as thrombocytopenia, anemia, opportunistic infections, and hyperviscosity . ChoriorRed eyes with subconjunctival nodules in patients with a history of previous AML should raise the suspicion for recurrent disease that warrants urgent biopsy and systemic treatment. It is suggested that eye relapse occurred because it may be an immune-privileged site such as the eyes, testis, and brain for AML relapse and will further highlight the importance of this site for cancer relapse. If there is a high suspicion for relapsed leukemia, systemic treatment is often integral to resolution of the ocular problems. It is usually responsive to systemic chemotherapy, local radiation treatment to the eye, or a combination of both of them.


In multicellular organisms, it plays fundamental roles during fertilization, development and adulthood. The inability of cells to regulate Ca2+ can lead to pathological conditions that ultimately culminate in cell death. One such pathological condition is manifested in Parkinson's disease, the second most common neurological disorder in humans, which is characterized by the aggregation of the protein, α-synuclein. This Review discusses current evidence that implicates Ca2+ in the pathogenesis of Parkinson's disease. Understanding the mechanisms by which Ca2+ signaling contributes to the progression of this disease will be crucial for the development of effective therapies to combat this devastating neurological condition.Across all kingdoms in the tree of life, calcium (Ca Summary: This Review article discusses the important roles that calcium plays in the pathogenesis of Parkinson's disease. Parkinson's disease (PD) is the second most common, multifactorial, progressive neurodegenerative disorder in humans after Alzheimer's disease, affecting 6.3 million people worldwide . CharactSNCA) is a major risk factor that is linked to sporadic PD  homeostasis . Oligomeric forms of the amyloid β (Aβ)-peptide, the major component of amyloid plaques , can create pores at the plasma membrane and trigger Ca2+-induced toxicity  is characterized by selective degeneration of motor neurons. The most compelling evidence linking Caal cells . Huntingm the ER .2+ can occur across the plasma membrane in response to electrical activity and/or through agonists. The electrical activity of neurons and other excitable cells relies on several different types of voltage- and ligand-gated ion channels that are permeable to inorganic ions, such as Na+, K+, Cl− and Ca2+. L-type  voltage-gated Ca2+ channels, Cav1.2 and Cav1.3, have been implicated in PD , or to the toxic effects of environmental factors known to cause PD by interfering with the mitochondrial complex I, namely rotenone or 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)  . In thesth in PD .Fig. 1.2+ channels in neurodegeneration opens up the possibility of repurposing these drugs to treat PD. Several epidemiological studies suggest that there is indeed a reduced risk of developing PD in patients with long-term use of Isradipine  to study the neuroprotective potential of Isradipine in early PD patients is currently ongoing and scheduled for completion in 2019   . Moreove channel  , Na+/Ca2+ exchanger (NCX), T-type Ca2+ channels at the plasma membrane, and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps, to reduce excitotoxicity and oxidative stress  reported an overall improvement in the patients' motor symptoms and a delay in symptom progression during the two and a half-year trial period , a member of the sialic acid-containing glycosphingolipids group that is highly expressed at the plasma membrane of neural cells, appears to have an important role in neuronal Cae stress . GM1 is e stress  and 2  responsible, as well as the mechanism behind this Ca2+ deregulation, have yet to be fully elucidated. Although the endoplasmic reticulum (ER), and to a lesser extent the mitochondria, are major intracellular Ca2+ stores, evidence suggests that other organelles, such as the lysosomes and Golgi, also act as important intracellular Ca2+ reservoirs . Interestingly, some PD-associated genes encode proteins that are involved in depleting Ca2+ from ER stores. For example, the gene BST1  is associated with sporadic PD in the European population (2+ release from the ER through the ryanodine receptors (RyR) by generation of cyclic ADP ribose (cADPR), a potent and universal Ca2+ mobilizer . Autosomal recessive mutations in PLA2G6 lead to early-onset dystonia-parkinsonism  and promotes refilling of the intracellular Ca2+ stores via activation of Ca2+ channels at the plasma membrane, a process called store operated Ca2+ entry (SOCE) -sensitive motor dysfunction in animal models  . PLA2G6 l models . In supprai1 see . These d2+ homeostasis can also have profound effects on other organelles through their physical connections. A good example of such interconnections is the ER-mitochondria contact sites, which form via mitochondria-associated membranes (MAM). As we discuss in the following section, these membranes are involved in several key processes, such as phospholipid and Ca2+ transfer, mitochondrial fission, mitophagy, the ER-stress response, and the regulation of apoptosis and inflammatory/antiviral responses  – and to changes in mitochondrial membrane permeability, both of which culminate in cell death  have been conducted in the last 10 years that have aimed at improving mitochondrial health during the course of the disease  complex in the inner mitochondrial membrane and with the inositol trisphosphate receptor (IP3R) on the ER. MCU and IP3R are coupled via the chaperone protein Grp75, which connects IP3R to the voltage-dependent anion channel type 1 (VDAC1) on the outer mitochondrial membrane  and the Na+/Ca2+ exchanger (mNCX), which are located on the inner mitochondrial membrane. Although the exact mechanism of action has yet to be established, two PD-associated proteins affect these mitochondrial Ca2+ import pathways: PINK1, by triggering the mNCX, and Parkin by stimulating VDAC1  was rescued in Gaucher patient-derived fibroblasts following treatment with the L-type Ca2+ channel blockers diltiazem or verapamil -dependent Ca2+ channels and members of the transient receptor potential channel superfamily, such as TPC (two-pore channels) and TRP   . The Ca2eostasis . Such a function , Fig. 1 function .ATP13A2 links Ca2+ homeostasis and lysosomal function to autosomal recessive juvenile onset of PD , which is important for vesicular transport from the endosomes to the Golgi, and the vacuolar protein sorting 13 (VPS13C), which is important for vesicular transport from the Golgi to the endosomes. These proteins affect Ca2+ influx in the secretory pathway and protein recycling , a neurodegenerative disease that shares some characteristics with PD , is a modifier of cytosolic Ca2+ and α-synuclein toxicity in yeast    (Chin anin (CaM) , 1993. Cproteins    , which has important roles in synaptic plasticity and long-term memory formation, and which is activated by phosphorylation and repressed in a calcineurin-dependent manner . As a sues (EOP) . EIF2AK3es (EOP) , Fig. 1 es (EOP) . EIF2AK3sy (PSP) . Althougsy (PSP) . In suppsy (PSP) . In addin the ER . Althoug2+-dependent group of enzymes implicated in PD are calpains. These cytosolic cysteine proteases are involved in the regulation of synaptic plasticity and long-term potentiation. Acute calpain activation is beneficial to neurons, whereas chronic activation induced by sustained cytosolic Ca2+ can lead to cell death. In support of a role for Ca2+ deregulation in PD, over-activated calpains have been detected in postmortem PD brains (CAST), which might predispose Caucasian individuals with European ancestry to idiopathic PD  and calbindin-D9k (encoded by S100G), are vitamin D-dependent Ca2+-binding proteins. Calbindin-D9k is mostly known for buffering Ca2+ in erythrocytes, whereas calbindin-D28k buffers Ca2+ in the central nervous system where it participates in the blockade of multiple pro-apoptotic pathways  is another Cal cortex , a regiol cortex . Alteredl cortex . Moreovel cortex . This su2+ signaling in biology, its involvement in the etiology of PD and other neurodegenerative disorders  and from intracellular stores . As such, understanding the mechanisms by which Ca2+ signaling contributes to the progression of PD is vitally important for developing effective therapies to treat this disease.Given the universal nature of Caisorders  might no2+ reservoirs. This suggests that Ca2+ could be a key player in coordinating complex organelle networks to ultimately achieve metabolic interactions, intracellular signaling, cellular maintenance and regulation of cell survival. Although neuronal cell culture models, in vivo rodent models and midbrain DA neurons derived from patient induced pluripotent stem cells (iPSCs) are vitally important tools for understanding the mechanisms underlying the pathology of PD, a significant investment of time and money is needed to make the most of these tools. Time is an issue when phenotypes are age dependent, as is the case for PD, and when the lifespan of rodents and/or primates is years. Even for iPSC-derived neurons, the time it takes in cell culture for any meaningful phenotype to appear can be up to a year. This, added to the high cost of performing mammalian-based experiments, makes these systems less than amenable for exploratory mechanistic research, despite the fact that they provide an essential validation tool for translation into the clinic. The use of model organisms such as yeast, flies and worms, can effectively circumvent these roadblocks. Indeed, these models have proven to be invaluable tools for uncovering conserved disease and cell biological processes that are affected in PD, which range from lipid biology, vesicular trafficking and function, lysosomal and peroxisomal function, autophagy, apoptosis, cell cycle, mitochondria and oxidative stress, Ca2+ signaling, ion channels and transporters, and the protein folding, quality control and degradation pathways. Model organisms can provide an excellent means to understand the mechanisms of Ca2+ deregulation in PD and could also shed light on how organelle networks operate to achieve cellular plasticity by using Ca2+ as a messenger, ultimately leading to novel therapeutic alternatives for combating PD.Most of the organelles affected in PD are major Ca


These observations provide a strong rationale for investigation of the specific effects of Suramin on skeletal muscle function. Single mechanically skinned fibers were directly exposed to Suramin  for defined durations (2–10 min) in controlled physiological solutions that mimic the intracellular ionic environment of a fiber. Suramin treatment (10–500 μmol L−1) directly affected the contractile apparatus in a dose‐dependent manner causing a decrease in Ca2+‐sensitivity (pCa50 = −log (Ca2+) concentration, where 50% of maximum Ca2+‐ activated force is produced) by 0.14 to 0.42 pCa units and reduction in maximum Ca2+‐activated force by 14 to 62%. Suramin treatment (100 μmol L−1 for 10 min and 500 μmol L−1 for 2 min) also caused development of a Ca2+‐independent force corresponding to 2.89 ± 4.33 and 16.77 ± 7.50% of pretreatment maximum Ca2+‐activated force, respectively. Suramin treatment  also increased the rate of sarcoplasmic reticulum (SR) Ca2+ release without significant changes in SR Ca2+ uptake. We report new functional effects for Suramin related to alterations in both the contractile apparatus and SR Ca2+‐handling of skeletal muscle that may contribute to the peripheral muscle weakness noted in human pharmacological treatments.Suramin has long been used in the treatment of various human diseases. Intravenous infusions of Suramin are commonly administered to patients over extended periods of time but there are a number of significant contraindications with peripheral muscle weakness being one of the most frequently reported. Previous work has shown that even after a single infusion (300 mg kg Due to global administration protocols and the broad acting nature of Suramin, a number of contraindications have been reported. Among the most significant side effects are severe fatigue, malaise and lethargy, which all may be manifest as a broad spectrum neuromuscular peripheral weakness of the hands, arms, legs and feet of patients  . Suramin has been shown to also affect SERCA . We show that Suramin treatment greatly affects both the Ca2+‐sensitivity and maximum Ca2+‐activated force and SR Ca2+ release through the RyR's. We hypothesize that as a result of Suramin treatment there will be changes in Ca2+‐sensitivity and maximum Ca2+‐activated force associated with direct effects on the contractile apparatus, as well as effects on the SR Ca2+ release through the RyR's. The combination of these actions helps to explain the Suramin‐induced peripheral muscle weakness noted in human pharmacological treatments.In this study, we use the mechanically‐skinned skeletal muscle fiber preparation  in accordance with the National Health and Medical Research Council (NHMRC) of Australia and ARRIVE guidelines.Rattus norvegicus), obtained from Monash University and housed at La Trobe University ranging in age from 215 to 324 days were used in these experiments. Animals were kept at the La Trobe University Animal House, housed 3 to a standard cage in a pathogen‐free environment, kept at a constant temperature (23°C) with 12 h day‐night cycles and provided with water and standard pellet rat chow ad libitum.A total of 24 male Long‐Evans hooded rats (−1). The extensor digitorum longus (EDL) muscle was dissected out immediately postmortem, blotted on filter paper and pinned under paraffin oil  at resting length on Sylgard 184 .Rats were killed by an overdose of isoflurane  fibers were isolated and mechanically skinned using a pair of fine forceps . As described by Fink and Stephenson , skinnedμm for all fibers) were then measured prior to application of a 20% stretch as previously described . All protocols utilized the force response of the skinned fiber segment as the primary measure of SR Ca2+‐buffered K‐EGTA solution (relaxing solution) which contained (mmol L−1): EGTA, 50 mmol L−1; HEPES, 90 mmol L−1; Mg2+total, 10.3 mmol L−1 ; ATPtotal, 8 mmol L−1; creatine phosphate (PCr)total, 10 mmol L−1; K+, 125 mmol L−1; Na+, 36 mmol L−1; pH, 7.10. Fibers were then exposed to a series of equivalent EGTA buffered solutions with progressively higher concentrations of free calcium (expressed in pCa units (−log[Ca2+]) from >9 to 4.5) by mixing different ratios of the relaxing solution above with a Ca2+‐EGTA solution (pCa 4.5) (maximum‐activating solution) that was identical in composition to the relaxing solution with the exception that it contained (mmol L−1): Mg2+total, 8.12 mmol L−1 ; Ca2+total, 49.7 mmol L−1. This resulted in the production of increasing force responses that plateaued once maximum Ca2+‐activated force was achieved at saturating free [Ca2+] resulting in the identification of the force‐Ca2+ relationship for the fiber . Once the maximum Ca2+‐activated force of fibers was confirmed, fibers were relaxed by re‐immersion in the relaxing solution. This activation sequence was repeated three times and fibers were only used if the maximum Ca2+‐activated force response in a sequence did not decrease by more than 5% between repetitions. Fibers were then incubated in a relaxing solution containing Suramin (100 μmol L−1 for 2 or 10 min or 500 μmol L−1 for 2 min). Fibers were then washed in fresh relaxing solution for 10 min to remove residual Suramin and the activation protocol was repeated three more times. The average of the force responses from the three activations was then used to construct a composite force‐pCa curve for each experimental group. For each of the three repetitions (both before and after Suramin treatment), the steady‐state submaximal force responses were normalized to its own maximum Ca2+‐activated force (considered 100%) and then the average of these three sets of values were used to construct a single composite force‐pCa curve which was represented graphically using GraphPad Prism v5.03. The pCa50 values (derived using equation 2+‐activated force was compared to the control (before treatment) force‐pCa relationship.Skinned fibers were immersed in a heavily CaY = Y‐axis; Bottom = Bottom Plateau; Top = Top Plateau; EC50 = X value corresponding to half‐maximal (Y) response; X = X‐Axis; HillSlope = Gradient at P50.where: 2+ and properties of SR Ca2+ release from the SR of skinned fibers was determined by examining the caffeine‐induced force response. The area under a caffeine‐induced force transient has been previously shown to be proportional to the SR Ca2+ content over a range of SR [Ca2+] (Endo and Iino −1): HEPES, 90; hexamethylenediamine‐tetraacetate (HDTA) 50; EGTA, 0.05; Mg2+total, 8.5 ; ATPtotal, 8; PCrtotal, 10; K+total, 125; Na+total, 36; pH 7.10) and fibers were bathed for 2 min and then briefly equilibrated in an identical solution , for 6 sec) before the SR was emptied of all releasable Ca2+ by exposure to a 30 mmol L−1 caffeine solution (30 mmol L−1 caffeine dissolved in a low (0.13 mmol L−1) [Mg2+free] equivalent of the K‐HDTA solution. This caffeine solution rapidly empties the SR of all stored Ca2+  for defined time periods  to precisely refill the SR with varying amounts of Ca2+. In between consecutive load periods, fibers were briefly washed (10 sec) in the wash solution, a process that stops further SR Ca2+ loading. Fibers were then transferred back to the 30 mmol L−1 caffeine solution triggering complete Ca2+ emptying of the SR. The ensuing area of the caffeine‐induced force transient was subsequently recorded. After the last loading period in the sequence described above (120 sec load), fibers were then washed for 12 min in the wash solution to account for the Suramin treatment time (see below) and then the SR was depleted again with 30 mmol L−1 caffeine solution to empty the SR of all Ca2+ before the load protocol (as above) was repeated for each loading period (10–120 sec) once more. This was done to test for reproducibility of the SR Ca2+‐ loading protocol over time. In separate fibers, this same procedure was repeated with the exception that after the initial control load curve was established, fibers were treated with 100 μmol L−1 Suramin (added to the wash solution) for 2 min followed by a further 10 min wash in a different wash solution to remove Suramin. A second load protocol was then repeated.A qualitative assessment of the amount of releasable Ca2+ by caffeine and any concomitant Suramin‐induced alterations in Ca2+ sensitivity and/or maximum Ca2+‐activated force (see below), it was necessary to estimate the profile of the Ca2+‐ change underlying the caffeine‐induced force response. The graphical relationship between force and the activating Ca2+ concentration represents a force‐pCa relationship as shown in Figure 2+‐force graph; Fig. 2+ concentration, this will account for the possibility that Suramin will affect the calcium sensitivity of each fiber and is required for each of the force transients generated by the pharmacological release  of SR Ca2+ of the same fiber using the following equationTo distinguish between the potential direct effects of Suramin on the pharmacological release of SR CaThis equation is a derivative of that previously devised by Rees and Stephenson n = average Hill coefficient (Hill slope) derived from corresponding force‐pCa curve; P/Pmax = Force (t)/Maximum Force (t) at each time point (t) of the force transient; pCa50 = Log of Calcium concentration at 50% of maximum force derived from the corresponding force‐pCa curve.where: 2+ traces by reference to the Ca2+‐force curve  peak free calcium concentration (μmol L−1) during caffeine‐induced Ca2+‐release from the SR, (2) maximum rate of Ca2+ rise (nmol ms−1) calculated as the rate of Ca2+ rise from the slope 20 to 80% of the peak Ca2+ transient, as an indicator of the rate of Ca2+‐release from the SR (the rates for different treatments were then expressed as a percentage of the 120 sec control) and (3) the Area Under the Curve of the resultant Ca2+ estimates associated with the caffeine‐induced Ca2+‐release as an indicator of total amount of SR Ca2+ content  was used for statistical analysis and curve fitting. Student t‐tests  were used to compare the means of the control and test groups. Lines of best fit to Ca2+ loading time‐dependent parameters were plotted using the following equation:Data are presented as means ± SD, and/or actual values where appropriate. Y=Ymax(1−A(−K×X)). Significance was indicated when P < 0.05 and denoted with an asterisk (*) for all statistical analysis where multiple analyses are carried out additional significance denoted with a hash (#).μmol L−1 Suramin for 2 min. Following a 2 min exposure to Suramin (500 μmol L−1) in a relaxing solution, and during the 10 min wash period in the “wash solution” (relaxing solution pCa >9) a pronounced Ca2+‐independent force developed. Subsequent determination of the force‐pCa relationship also revealed a marked decrease in the Ca2+‐activated force across the entire pCa range.Figure 2+ concentration in a single fiber treated with 10 μmol L−1 for 2 min is presented in Figure 2+‐Active Force and “Resting” Force [force in relaxing solution of pCa > 9]) for the four variations of Suramin treatment shown. All four treatment groups showed a statistically significant decrease in Ca2+‐sensitivity . Fibers treated with 10 μmol L−1 Suramin showed a significant change in pCa50 with an average decrease in 0.14 ± 0.11 (n = 5). The group treated with 100 μmol L−1 Suramin for 2 min also had a significant change in the pCa50 value with an average decrease in 0.18 ± 0.09 (n = 9). Fibers treated with 100 μmol L−1 Suramin for 10 min in addition to an average decrease in the pCa50 of 0.36 ± 0.19 also showed a decrease in the Hill slope co‐efficient of 2.67 ± 0.76 (n = 10) and the presence of Ca2+‐independent force (~3% of the maximum Ca2+‐activated force). Fibers treated with 500 μmol L−1 Suramin (2 min) showed further significant decreases in both pCa50 and the Hill slope co‐efficient of 0.42 ± 0.13 and 1.11 ± 0.83 (n = 11), respectively, and the presence of considerable Ca2+‐independent force of ~16% of the maximum Ca2+‐activated force.The effect of Suramin on the graphical relationship between force and the activating Ca2+‐activated force was also significantly affected by Suramin treatment (Table μmol L−1 Suramin (2 min) showed a decrease in the maximum force by 14 ± 7.16% compared to the control. This decreased further by 18.9 ± 15.96% in fibers treated with 100 μmol L−1 Suramin (2 min) and by 37.4 ± 38.93% after a 10‐min treatment. Muscle fibers treated with 500 μmol L−1 Suramin for 2 min exhibited the greatest reduction in maximum active force with an average reduction in 61.8 ± 13.83%. These data reveal that Suramin has a significant effect on both the Ca2+‐activated force and Ca2+‐independent (resting) force exerted through a direct effect on the contractile apparatus. The significant reduction in both maximum Ca2+‐activated force and the development of Ca2+‐independent force after Suramin treatment, suggests that Suramin potentially removes key regulatory proteins of the contractile apparatus – the troponin complex.The maximum Cant Table . Fibers 2+‐dependent and independent force observed after Suramin treatment are due to removal of the Troponin complex, a subset of Suramin treated fibers were immersed in a relaxing solution that contained equimolar concentrations (5 mmol L−1) of Troponin I, Troponin T and Troponin C rabbit proteins after we first checked on three control fibers that such troponin treatment did not affect the force responses in these fibers.To test whether the changes in both Caμmol L−1, 10 min treatment, 10 min wash and 500 μmol L−1 Suramin, 2 min treatment and 10 min wash). Troponin re‐addition did not reverse the changes in Ca2+ sensitivity caused by Suramin (100 or 500 μmol L−1), but was able to reverse a significant component of the reduction in the Hill slope of the force‐pCa curve caused by 100 μmol L−1 Suramin application. Similarly, troponin re‐addition partially restored the diminished capacity of fibers to generate Ca2+ activated force following 100 μmol L−1 Suramin treatment and significantly reduced the Ca2+‐independent force generated by both Suramin treatments by ~50%.Table 2+ transients as indicated in the Methods  of the free Ca2+ transients for control and Suramin treated fibers, respectively, C & D show the rate of Ca2+ rise (% nmol ms−1) called “rate of release” measured between the period 20 through to 80% of peak, and E & F show the area under the free Ca2+ transient  produced a significantly higher peak Ca2+‐transient at all loading times compared with the preceding control. Suramin caused an increase in the peak Ca2+‐transient by approximately twofold across the entire load curve.Figure 2+ release from the SR under control conditions significantly increased by a small amount after the 12 min control wash period  compared to the control.The rate of Caiod Fig. C. Howeve2+‐transient described above, which are affected directly by any change in RyR channel activity, the relative area under the caffeine‐induced Ca2+‐transient provides (see 2+ loaded into the SR and thus an indication of the activity of the SR Ca2+‐ATPase (SERCA) and the total amount of Ca2+ subsequently released from the SR. Using the equation Y=Ymax(1−A(−K×X)) to plot a line of best fit through all loading times, we could assess the amount of Ca2+ loaded into the SR in control and Suramin treated fibers  the maximum Ca2+‐activated force elicited at pCa 4.5 (see Table 2+ decreased by 38% (δpCa50 = −0.14). Furthermore, at higher Suramin concentrations (100–500 μmol L−1 Suramin), the maximum Ca2+‐activated force was decreased by 62%, the Ca2+‐sensitivity was decreased by a factor of 2.3–2.6 (δpCa50 = −0.36 to −0.42), and in addition, treated fibers developed a Ca2+‐independent force , partially reversed the effects of Suramin observed on resting force, maximum force and Hill slope. This suggested that the effects of Suramin on the contractile apparatus were partially mediated through the removal of Troponin C and Troponin I. Removal of Troponin C is consistent with the reduction in the Ca2+‐activated force. Given that Suramin has previously been reported to remove CaM from the RyRs  force which would contribute to an elevated resting cellular ATPase activity that would further lead to a generalized metabolic fatigue.This study demonstrates that Suramin does not only contribute to muscle weakness by affecting the properties of the RyRs but also has a strong lasting effect on skeletal muscle myofibrillar components which markedly reduces the ability of muscle to produce CaNone declared.


Background. Nitric oxide can successfully compete with oxygen for sites of electron-transport chain in conditions of myocardial hypoxia. These features may prevent excessive oxidative stress occurring in cardiomyocytes during sudden hypoxia-reoxygenation. Aim. To study the action of the potent stable NO donor dinitrosyl iron complex with glutathione (Oxacom®) on the recovery of myocardial contractile function and Ca2+ transients in cardiomyocytes during hypoxia-reoxygenation. Results. The isolated rat hearts were subjected to 30 min hypoxia followed by 30 min reoxygenation. The presence of 30 nM Oxacom in hypoxic perfusate reduced myocardial contracture and improved recovery of left ventricular developed pressure partly due to elimination of cardiac arrhythmias. The same Oxacom concentration limited reactive oxygen species generation in hypoxic cardiomyocytes and increased the viability of isolated cardiomyocytes during hypoxia from 12 to 52% and after reoxygenation from 0 to 40%. Oxacom prevented hypoxia-induced elevation of diastolic Ca2+ level and eliminated Ca2+ transport alterations manifested by slow Ca2+ removal from the sarcoplasm and delay in cardiomyocyte relaxation. Conclusion. The potent stable NO donor preserved cardiomyocyte integrity and improved functional recovery at hypoxia-reoxygenation both in the isolated heart and in cardiomyocytes mainly due to preservation of Ca2+ transport. Oxacom demonstrates potential for cardioprotection during hypoxia-reoxygenation. Also, the activity of Ca2+-ATPase of sarcoplasmic reticulum (SERCA2) declines quite rapidly [2+ uptake in the reticulum and subsequent release into sarcoplasm. However, an increased level of sarcoplasmic Ca2+ in diastole [Oxidative stress in cells occurs when reactive oxygen species (ROS) prevail over the means of antioxidant protection. Reoxygenation after a period of hypoxia is a classic model of oxidative stress. Cardiomyocytes in hypoxic conditions mobilize a number of protective mechanisms to enable them to retain integrity in this critical period and restore normal function at reoxygenation. A deep fall in contractile function allows them to conserve limited stores of ATP and phosphocreatine. This is realized by means of limiting Cato cells  due to tdiastole , especiadiastole  with the2+ complexes in the presence of glutathione [In the last decade, many aspects of altered redox regulation in hypoxia became more evident. Although oxygen consumption during hypoxia is reduced, the mitochondria continue to generate superoxide , althougtathione .In light of these findings, elevated NO levels could potentially be useful during reoxygenation when the availability of oxygen to mitochondria suddenly multiplies. The well-known NO donors such as nitroglycerin, nitrosorbide, nitroprusside, and nitrite liberate NO in cells and tissues during their metabolism. However, some of these substances, such as sodium nitroprusside, contain cyanide anions, while other compounds including nitroglycerin can induce methemoglobinemia. As a result of decades-long research of professor Anatoly F. Vanin  and his colleagues, it was established that S-nitrosothiols present in cells can easily interact with iron ions released from the iron donor ferritin to form dinitrosyl iron complexes (DNIC) , 11. TheThis concept was embodied at the Russian Cardiology Research and Production Complex by creating a drug Oxacom (Patent RU2291880) which represents a DNIC with glutathione as a ligand. In preclinical studies, a dose-dependent hypotensive effect of Oxacom in conscious rats was observed . Oxacom n = 48) anesthetized with ketamine (100 mg/kg). The protocol of experiments was approved by the Animal Ethics Committee of the Institute of Experimental Cardiology of the Russian Cardiology Research and Production Complex (# 3/15). Hearts were isolated from rats anesthetized with ketamine (100 mg/kg). Hearts were cannulated and coronary vessels were retrogradely perfused at 37°C using standard Krebs-Henseleit solution containing 11 mM glucose and presaturated with carbogen . A latex balloon was introduced into the left ventricle (LV) and isovolumic pressure in LV and ECG were monitored. The balloon was filled with liquid to set diastolic pressure at 10–12 mm Hg in order to achieve an optimal distension of LV. Retrograde perfusion was conducted at constant pressure of 70 mm Hg. Electrodes were placed on the right atrium for electrostimulation.Hearts were isolated from male Wistar rats (dP/dt divided by LV developed pressure) were calculated.Heart perfusion was performed in a specialized Hugo Saks perfusion system. The LV pressure and ECG were recorded using Harvard Apparatus (USA) sensors and amplifiers. All signals (LV pressure and coronary perfusion rate) were transferred to the computer through the preliminary amplifiers and analog-to-digital converter . In addition to analog signals  the heart work index (the product of developed pressure and heart rate), which characterizes the overall energy consumption, and relaxation index  using averaging intervals from 6 s to 30 min; (b) linking the beginning of an averaged segment to the mark introduced by the operator during the experiment; (c) averaging between marks; and (d) combining averaging modes. The results of calculations can be written as text files or directly transferred to Excel spreadsheet using a ClipBoard function.2 by N2 in perfusate) was followed by 30 min reoxygenation. This protocol was chosen after the preliminary tests, which revealed that 20-minute hypoxia was insufficient to cause cardiac dysfunction and the degree of recovery was close to 100%. At the end of experiment, electric stimulation with increasing rate was repeated. Oxacom (30 nM) was added to perfusate during hypoxic perfusion and it was not added at reoxygenation.After recording of the cardiac baseline function, stepwise electric stimulation with increasing rate exceeding the spontaneous rate was applied using increments of 0.5 Hz up to 12 Hz and pacing for 15 sec at each rate. Then electrostimulation was stopped and 10 minutes later the functional parameters were recorded before the start of hypoxic perfusion. The 30 min hypoxia  was used for measurements of total glutathione (GSH + GSSG) content using Glutathione Assay Kit (Sigma) according to the manufacturer's instructions in a buffer containing 95 mM KH2PO4, pH 7.0, 0.95 mM EGTA, 48 mM NADPH, 0.031 mg/mL 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 0.115 U/mL glutathione reductase, and 0.24% SSA in a final volume of 200 μL. Kinetics of DTNB reduction to 5-thio-2-nitrobenzoic acid (TNB) were measured using a Victor X3 plate reader (Perkin Elmer) at 405 nm. The amount of glutathione was calculated using a standard curve of reduced glutathione. Data were expressed as a mean from at least triplicate measurements ± standard deviation (SD). Amount of reduced glutathione (GSH) was calculated from measurements where both NADPH and glutathione reductase were excluded from the assay mixture. Average relative amount of reduced glutathione in the heart was calculated based on determined values.Rat hearts were washed with phosphate buffered saline and frozen in liquid nitrogen immediately after experimental procedures. The tissue was ground in liquid nitrogen, and three volumes of 5% 5-sulphosalicylic acid (SSA) were added to 0.1–0.3 mg of the powder. The tissue sample was extracted using a glass-glass homogenizer on ice and centrifuged at 10,000 ×g for 10 min at 4°C to pellet the proteins. The supernatant fraction  for 30 min. Rat heart was cannulated through aorta and perfused for 10 min according to the method of Langendorf to remove blood with Krebs-Henseleit buffer supplemented with 1.2 mM Ca2+. Next, heart was perfused with Ca2+-free buffer for 30 min to remove Ca2+ from extracellular spaces. After washing steps, heart was perfused in a recirculation mode for 30 min with 10 mL of 0.5 mg/mL collagenase  solution in a Ca2+-free buffer. Following perfusion the heart was placed in a petri dish with 10 mL of fresh collagenase solution and minced in 1-2 mm3 pieces using fine scissors. Tissue suspension was transferred in 50 mL tube and gently pipetted for 10–15 min using sterile 5 mL plastic pipette with wide opening. Digested tissue was filtered through the 200 micron Nylon mesh to separate isolated cardiomyocytes from undigested heart fragments. Cardiomyocyte suspension was left in a sterile 50 mL tube for 10 min to settle down live cells. Then the supernatant was removed and cardiomyocytes were gently resuspended in 10 mL of albumin solution (6 mg/mL) supplemented with 0.1 mM Ca2+ and left for 10 min to settle. This step was repeated several times by placing cells in an albumin solution with increasing concentration of Ca2+ using steps of 0.25 mM until 1 mM Ca2+ was set. Cardiomyocytes were kept at 4°C in Krebs-Henseleit solution supplemented with 1 mM Ca2+ and 6 mg/mL albumin no more than 12 hours after isolation.Right before cardiomyocyte isolation all buffers were supplemented with 1000 U/L heparin, warmed to 37°C in a water bath, and bubbled with carbogen , respectively, and the chamber space above the buffer was constantly flashed with the corresponding gas mixture.In order to reproduce normoxic or hypoxic conditions during the experiment cardiomyocytes were placed in experimental chamber in the buffer bubbled with carbogen or hypoxic gas mixture (95% N2+ indicator Fluo-4 (Invitrogen) for 20 min in the dark and transferred in a 250 μL experimental chamber made of plastic walls and glass coverslip as the bottom. Two platinum electrodes are fixed at the opposite walls of the chamber. These electrodes are connected to Grass SD9 electrostimulator and are used to pace cardiomyocytes in the chamber by rectangular electric impulses at 38 V and 1 Hz. The chamber is mounted on the stage of an inverted AxioVert 200 M microscope (Zeiss) in a thermostat set at 37°C. Buffer is delivered in the chamber by gravity flow at 1–5 mL/min and removed from the chamber using a peristaltic pump. Cardiomyocytes are monitored through the coverslip using x63 oil objective and AxioCam HS high speed CCD camera (Zeiss). Fluorescence of Fluo-4 is excited using an HBO 103W/2 mercury lamp (Osram) and an appropriate filter cube for FITC-based fluorophores. Illumination intensity is kept low and exposure times are made short in order to prevent Fluo-4 bleaching and cardiomyocyte damage. Fluorescent signal from the individual cardiomyocytes is recorded at 50–200 frames/sec and images are streamlined to a terabyte hard drive for further processing using AxioVision Physiology software (Zeiss).Cardiomyocytes were loaded with fluorescent Ca2+-transient measurements in cardiomyocytes. Cells were loaded with DHR 123 for 20 min. Electrostimulation of cardiomyocytes was done in parallel with fluorescence recording and between interventions cells were kept in the dark.Fluorescent indicator Dihydrorhodamine 123 (Thermo Fisher Scientific) was used to assess reactive oxygen species (ROS) accumulation in isolated rat cardiomyocytes. The same experimental setup was used as described above for Cat-test. Values in the tables and text are given as M ± SEM unless indicated differently.Statistical processing of experimental data obtained in isolated heart and isolated cardiomyocyte studies was performed using Statistics Package in Microsoft Excel 2013 and Student's μM in oxygenated hearts. The coronary flow rate was monitored as the main parameter as the increased rate under constant perfusion pressure indicates a decreased tone of the coronary vessels. Coronary flow rate clearly increased from 16.0 ± 1.2 to 20.2 ± 1.2 mL/min, but the changes in LV developed pressure were modest, about +7–9%. Only Oxacom concentration of 2.7 μM caused a distinct decrease in LV developed pressure by 24%. In this regard, for further work the concentration of Oxacom 30 nM was selected, which reliably increased coronary flow rate and the index of relaxation by 8-9%. Both these effects could be interpreted as facilitation of Ca2+ removal from smooth muscle cells and cardiomyocytes, the events compatible with the reduction of hypoxic injury to myocardium.In order to determine an optimal concentration of Oxacom to be used during hypoxic perfusion, we studied this compound across the concentration range of 0.01–2.7 Baseline cardiac functional parameters are important for recovery of function after hypoxia-reoxygenation. It is known that lower intensity of oxidative metabolism at the beginning of hypoxia provides better recovery after reoxygenation. In this regard, the experiments where LV developed pressure was below 130 mm Hg and heart rate was below 230 beats/min were excluded from analysis.Hypoxic perfusion of the heart resulted in a steep drop of LV developed pressure already in the first minutes of hypoxia along with increasing LV diastolic pressure that characterized myocardial contracture. Cardiac arrhythmias were observed in hypoxic period and they increased dramatically during reoxygenation. Arrhythmic events included extrasystoles, idioventricular rhythm, and fibrillation, which ceased spontaneously. Altogether, due to these reasons 4 hearts of 15 total did not restore function at the end of experiments.By the end of reoxygenation the heart rate restored function completely while LV developed pressure restored function only by 27 ± 6% (by 34 ± 4% excluding experiments with zero recovery). The cardiac work changed accordingly. The LV diastolic pressure dramatically rose during hypoxia and slightly increased by the end of reoxygenation averaging 57 ± 5 mm Hg.During 10–30 min period of hypoxia in the presence of Oxacom the plateau level of LV diastolic pressure was consistently lower by 12–20 mm Hg than in control and this difference was statistically significant . During dP/dtmax parameter was significantly higher than in control.In both groups studied, the cardiac functional parameters before hypoxia were similar . After hp < 0.02).Recurrent arrhythmias were observed during both periods of hypoxia and reoxygenation in control and Oxacom experiments. Overall, the arrhythmias were observed in 10 out of 11 experiments in control group and in 7 out of 9 experiments in Oxacom group. The average integral length of arrhythmic events in control group was 14 ± 2 min, half of which originated from ventricular tachycardia (7 ± 1 min). In Oxacom group, the same distribution of arrhythmic events was observed; however, the values were significantly smaller, 4 ± 1 min and 2 ± 1 min . Later p < 0.01), 34 ± 6% at 20 min , and 41 ± 3% at 30 min .The time course of LV developed pressure highly depended on arrhythmias because, during arrhythmic episodes, it dropped almost to zero. Still, in the presence of Oxacom LV pressure was maintained at higher levels throughout the hypoxic period and especially at the start of reoxygenation when arrhythmias in Oxacom group quickly ceased . The devAn attempt to restore cardiac function using L-arginine was undertaken by Agulló et al. . They adElectrostimulation with increasing rate was performed before and after hypoxia-reoxygenation. The results shown in p < 0.02). Further increase of stimulation rate resulted in the failure of 3 out of 11 control hearts to reproduce the frequency above 10 Hz while two hearts failed at 8 Hz. In contrast, all the hearts in Oxacom group successfully reproduced the highest applied stimulation rate. Based on the heart performance, the average cardiac work index in Oxacom group was 1.5–2-fold higher than in control group (p < 0.01). At maximal rate of stimulation, the cardiac work index in Oxacom group accounted for approximately 75% of the CWI for untreated normoxic heart.After reoxygenation cardiac work index in control group stimulated at initial 5 Hz rate was significantly lower than in Oxacom group  and Ca-ATPases such as SERCA2 and PMCA in cardiomyocytes. Working in direct mode NCX expels one Ca2+ ion from the sarcoplasm into extracellular space in exchange for three Na+ ions using electrochemical gradient for Na+ across sarcolemma generated by Na/K-ATPase. NCX has low affinity and high capacity for Ca2+ and is mostly active at peak Ca2+ concentration in the sarcoplasm. SERCA2 and PMCA have higher affinity and lower capacity for sarcoplasmic Ca2+ and are more effective at moderate to low Ca2+ concentrations.Control cardiomyocytes electrically paced at 1 Hz under normoxic conditions respond by the regular Camulation . The sha2+ from the sarcoplasm to achieve its basal levels before the next electric stimulus arrives. The total length of Ca2+ transient under control normoxic conditions is 200–250 ms. During hypoxia the peak levels of free ionized Ca2+ entering cardiomyocytes modestly increase judging by the increase of Ca2+ transient amplitude  the average content of free ionized Ca2+ per Ca2+ peak is 3-fold higher in hypoxic than in normoxic cardiomyocytes indicating substantial Ca2+ overload. Noteworthily, in our experimental model cardiomyocytes hardly recover from more than 2 min of hypoxia and subsequent reoxygenation after this period of time does not improve their Ca2+ transients indicating irreversible damage to the molecular systems of Ca2+ transport . However, such low dose of Oxacom was not effective in isolated perfused heart model.In contrast, these negative events are not developed when cardiomyocytes are preincubated with 30 nM Oxacom and this compound is present in the perfusate throughout the experiment Figures . Oxacom 2+ transport in these stressful conditions.Thus, Oxacom prevents alterations induced by hypoxia-reoxygenation in Ca-ATPases and, perhaps, NCX and allows cardiomyocytes maintain normal Ca2+-transport; each Ca2+ peak was followed by a single contraction of cellular body. Prolonged Ca2+ elevations in hypoxia were accompanied by the same lengthy cellular contractions. Thus, the effect of Oxacom on contractile activity of cardiomyocytes in our model was mainly determined by its impact on the process of Ca2+ removal from the sarcoplasm.Contractile activity of cardiomyocytes, based on their visual assessment under the microscope, was fully consistent with the parameters of CaOxacom improved viability of cardiomyocytes subjected to hypoxia-reoxygenation. Our isolation procedure typically yields about half of viable cardiomyocytes in the total cell isolate. These cells display characteristic elongated shape with sharp edges . After tExposure of cardiomyocytes to 2 min hypoxia in the presence of Oxacom resulted in 52% elongated cells in the sample  similar The alterations of ion-transporting molecular systems of cardiomyocytes in conditions of hypoxia-reoxygenation and ischemia-reperfusion are often linked to the damage of ion transporters and regulatory proteins by reactive oxygen species (ROS) that accumulate in cells during the disturbances in oxygen supply. As shown in However, both hypoxia and electrostimulation lead to increased ROS in control cells. Reoxygenation initially increases the levels of ROS in cardiomyocytes whereas about 1 min after the start of reoxygenation further increase in ROS is blunted regardless of cardiomyocyte electrostimulation. Perhaps, this reflects the recovery of the normal redox state in cardiomyocytes following oxygen availability. Overall increase in ROS achieved in control cardiomyocytes during hypoxia-reoxygenation in our experiments was about 3.5-fold.In the presence of 30 nM Oxacom cardiomyocytes demonstrated no significant increase of ROS compared to control cells when subjected to hypoxia/electrostimulation. Specifically, Oxacom attenuated the accumulation of ROS during the period of hypoxia 6-fold. Similarly, during the first minute of reoxygenation Oxacom limited ROS accumulation; however, noticeable increase in ROS was observed at a later time with no evidence for saturation. Overall increase in ROS was about 2-fold in cardiomyocytes treated with Oxacom, which is 75% less than in control cardiomyocytes. Thus, in the presence of Oxacom the accumulation of ROS in cardiomyocytes during hypoxia was markedly attenuated and the latter effect could be related to the better performance of Ca-ATPases and, perhaps, NCX/Na/K-ATPase in these cells.2+-transporters in reduced state and/or protects them through a reversible S-nitrosylation. It is known that cysteine residues of proteins located on the cytosolic side of sarcoplasmic reticulum membrane [2+-channels, and L-Type Ca2+-channels [2+ transporting proteins [2+-channel activity was demonstrated [It is possible that Oxacom acts to maintain the thiol groups of SERCA2 and other Camembrane  may undechannels , 22. Becproteins . In the nstrated . Modific2+ accumulation in hypoxic cardiomyocytes favor NO signaling pathway rather than the direct nitrosylation of the set of cellular proteins. Finally, the attenuation by Oxacom of ROS generation during the period of hypoxia suggests possible stimulatory effects of this compound on antioxidant defense systems of cardiomyocytes different from glutathione system. One possible scenario is that by direct scavenging of the hydroxyl and superoxide radicals NO could protect cardiac Na/K-ATPase from inactivation [2+ overload through NCX working in reverse mode. Outlined multiple possibilities warrant further studies to elucidate in detail the molecular basis of Oxacom action on Ca2+ transporters and ROS production; such experiments have already been initiated.Based on the current estimations of in vivo nitric oxide levels ranging from 100 pM or less up to 5 nM  one can tivation  and cons2+ removal from the sarcoplasm and delayed cardiomyocyte relaxation.Obtained results demonstrate that Oxacom is an effective pharmacologic compound capable of reducing hypoxic contractures and promoting increase in the speed and extent of LV developed pressure recovery during reoxygenation. Its action to attenuate cardiac arrhythmias and to preserve the ability of the heart to reproduce high rate of contractions suggests that Oxacom can improve the function of calcium transport proteins in cardiomyocytes. This assumption was supported by direct experiments in isolated cardiomyocytes. Oxacom eliminated calcium transport alterations that took place in control experiments in the form of the slow Ca2+ removal from the sarcoplasm by Ca2+-ATPases of the sarcolemma and sarcoplasmic reticulum (PMCA and SERCA2) as well as by NCX/Na/K-ATPase tandem is energy-dependent process that slows down due to the depletion of high-energy phosphates during the shortage of oxygen. However, contractile process requires much more energy than the work of the membrane ion pumps. In this regard, the prevention by Oxacom of hypoxic contracture and more rapid recovery of the contractile function during reoxygenation in the presence of this compound suggest protective effects on mitochondria. Observed action of Oxacom to limit ROS generation in hypoxic cardiomyocytes additionally supports the mitochondrial vector for the future mechanistic studies of this novel cardiovascular drug.This corresponds well to the view that Ca2+ transients in rat cardiomyocytes subjected to hypoxia-reoxygenation in the presence of 0.3 nM Oxacom.Experimental data on Ca


P < 0.01). Rabbits with diarrhea had significantly lower concentrations of acetic acid (P < 0.05), higher pH levels (P < 0.05), and higher levels of ammonia nitrogen (P < 0.01) in the cecum. Moreover, diarrhea in baby rabbits led to significantly reduced levels of total serum protein (P < 0.05) and markedly increased levels of alkaline phosphatase, urea nitrogen, TNF-α, and IL-6 (P < 0.05). Transcriptional analysis of peptidoglycan recognition proteins  using real-time PCR revealed that diarrhea induced the upregulation of PGRPs in the cecum and duodenum. Furthermore, through pyrosequencing of the 16S rRNA V4 region in cecum samples, we found that the total number and diversity of microbes were not significantly different between healthy rabbits and those with diarrhea, though there were noticeable differences in the prevalences of Clostridium, Roseburia, and Alistipes. Our results will contribute to a better understanding of the pathological mechanisms of diarrhea in young rabbits.Diarrhea is a syndrome of digestive disorders in young rabbits and may lead to secondary infections resulting in reduced immunity and higher mortality in baby rabbits, with serious impacts on rabbit farming. In this study, we investigated the effects of diarrhea on the health of baby rabbits in terms of intestinal mucosal development, immune function, and intestinal microbial diversity. We found that the duodenal villus length and the villus/crypt ratio in rabbits with diarrhea were significantly reduced compared with those in healthy rabbits ( Diarrhea is a common disease in rabbits, especially in weaned baby rabbits, in which it has the highest incidence among all diseases . It caus9–1010Bacteroides in every gram of cecal content in an adult rabbit, whereas the total amount of Bifidobacteria, Clostridium, Streptococcus, and Enterobacter is 1010–1012 CFU/g  and were strictly implemented according to the regulations for experimental animals. An ordinary housing facility was used and was in keeping with the national standard Laboratory Animal Requirements of Environment and Housing Facilities (GB 14925-2001). The care of laboratory animals and the animal experimental operations conformed to the Jiangsu Administration Rule of Laboratory Animal. The rabbits were anesthetized by an intraperitoneal injection of sodium pentobarbital.3-N) levels, as well as 16S high-throughput sequencing. The duodenum and cecum samples were collected at the same position in each rabbit and stored in liquid nitrogen for later RNA extraction. A fragment of the duodenum was removed at the same position, rinsed with 0.9% saline, and fixed in paraformaldehyde for the preparation of intestinal sections.New Zealand rabbits weaned at 35 days of age were used as experimental animals to study the impacts of diarrhea. Rabbits with spontaneous diarrhea were collected 10 days after weaning. Moreover, eight weaned rabbits with diarrhea and eight healthy weaned rabbits were obtained and sacrificed. The cecal contents of each rabbit were collected in three 2-mL cryotubes and stored in liquid nitrogen for later determination of VFA and ammonia nitrogen . Duodenal contents (2 mL) were mixed with 2 mL distilled water and recovered by centrifugation at 10,000 rpm for 10 min. Subsequently, 1 mL supernatant was collected and mixed with 0.2 mL 20% metaphosphoric acid solution containing 60 mM crotonic acid. VFA levels were determined using a GC-9A Gas Chromatograph  with nitrogen as the carrier gas, a flow-rate of 30 mL/min. A CP-WAX capillary column with a length of 30 m, an inner diameter of 0.53 mm, and a membrane thickness of 1 µm was used, and the sample input was 0.6 µL.3-N in the cecum was analyzed based on a standard curve of ammonium chloride.Diluted cecal contents (4 mL) were centrifuged for 10 min, and 50 µL supernatant was collected in a 10-mL tube. Phenol and sodium hypochlorite (3 mL each) were added to the supernatant, and the mixture was incubated in a 60 °C water bath for 10 min, followed by immediate cooling. The optical density (OD) at 546 nm was measured, and the concentration of NHα (TNF-α), immunoglobulin G (IgG), and immunoglobulin M (IgM), reflected the immune response to diarrhea.Blood was drawn from rabbits with diarrhea and healthy rabbits, allowed to settle for 30 min, and centrifuged at 3,500 rpm for 15 min to isolate serum. Serum indicators were detected according to the instructions of the manufacturer . The factors used to evaluate the serum biochemical profile included serum glucose (GLU), total protein (TP), urea nitrogen (UN), and serum alkaline phosphatase (AKP). In particular, serum immune indicators, including interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor-α, IgG, and IgM were determined by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).The level of GLU was measured using the glucose oxidase method. TP content in the serum was determined by the biuret method. The level of UN in the serum was determined using the urease method. AKP was measured by colorimetry. The serum levels of IL-2, IL-6, TNF-The intestine was examined by routine HE staining of tissue paraffin sections. Five consecutive longitudinal sections were obtained from each segment of the intestinal tract, and six typical microscopic fields were assessed for each tissue section. The lengths of the longest villus and the corresponding crypt in each field were measured. The intestinal wall, villi, and crypt are shown in GAPDH expression using the SYBR Premix Ex Taq II system . Based on the mRNA sequences of rabbit PGRPs published in GenBank, real-time PCR primers for PGLYRP-1, PGLYRP-2, and PGLYRP-3 were designed and are shown in Ea is the amplification efficiency of the target gene, Ek is the amplification efficiency of the internal control gene, CTca is the Ct value of the target gene in the control group, CTta is the Ct value of the target gene in the test group, CTck is the Ct value of the internal control gene in the control group, and CTtk is the Ct value of the internal control gene in the test group.Real-time PCR was carried out on Applied Biosystems 7500 Real-Time RT-PCR System with the following program: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. All cDNA samples were tested three times, and the results were normalized to the levels of t-tests for independent samples and one-way ANOVA (SPSS version 21). The results are presented as the mean ± SD at two levels of significance, P < 0.05 and P < 0.01.Each experiment was repeated at least three times, and the data were analyzed using Homogenized cecal contents weighing 3 g (wet weight) from each healthy rabbit and rabbit with diarrhea were used (three rabbits from each group) for DNA extraction. Genomic DNA was extracted using hexadecyl trimethyl ammonium bromide (CTAB). After testing for DNA purity and concentration, an appropriate amount of sample was diluted with sterile water to a final concentration of 1 ng/µL.The 16S V4 amplicon was amplified by PCR using specific barcode-containing primers . The PCR products were mixed in equal quantities based on their concentrations, and the mixture was subjected to electrophoresis on a 2% agarose gel (1 × TAE) for the purification of PCR products. The major bands at 400–450 bp were collected using GeneJET (Thermo Scientific). Thereafter, the library was constructed using the TruSeq DNA PCR-Free Library Preparation Kit , and the quality of the library was assessed using a Qubit® 2.0 Fluorometer and Agilent Bioanalyzer 2100 system. After quality testing, libraries were sequenced by Illumina HiSeq.SRP114741).We merged paired-end reads from the original DNA fragments and applied quality filters using FLASH . The resP < 0.01), as well as significantly thinner duodenal walls (P < 0.05). However, crypt depth was not significantly different between the two groups , but the concentrations of propionic acid and butyric acid as well as the ratio of acetic acid/(propionic acid + butyric acid) were comparable to those in the healthy group (P > 0.05). Moreover, diarrhea led to markedly increased pH (P < 0.05) and NH3-N content (P < 0.01). Under normal conditions, propagating probiotics produce lactic acid and acetic acid, reducing the pH in the digestive tract and modulating the microenvironment to inhibit the growth of harmful bacteria.The characteristics of the cecal environment are shown in P < 0.05), whereas AKP, UN, TNF-α, and IL-6 were significantly higher in the diarrhea group than the healthy group (P < 0.05). Levels of GLU, IgG, IgM, and IL-2 were not significantly different between the two groups (P > 0.05). These results suggest that protein breakdown and consumption increases in diarrhea, thus impairing body development.Changes in serum indicators after diarrhea were examined. As shown in PGLYRP-1 and PGLYRP-2 were both significantly upregulated in the ceca and duodena of rabbits with diarrhea compared with levels in healthy rabbits (P < 0.05), while PGLYRP-3 was significantly upregulated in the ceca of rabbits with diarrhea (P < 0.05) but not significantly altered in the duodena (P > 0.05). These data suggest that young rabbits might combat harmful, diarrhea-causing intestinal bacteria by upregulating intestinal PGRPs.Compared with the healthy group, the expression of antibacterial PGRPs increased in the ceca and duodena of rabbits with diarrhea . PGLYRP-P > 0.05). To assess microbial diversity, clustering of effective tags was performed on all samples by sorting tags with ≥97% identity into an OTU. There were 916 ± 28 and 880 ± 46 OTUs in the healthy group and the diarrhea group, respectively, implying a higher diversity of cecal flora in healthy New Zealand rabbits. However, floral diversity was not significantly different between the diarrhea and healthy groups (P > 0.05)  tags were detected in each sample, with no significant difference between the healthy  and diarrhea groups  ( > 0.05) .The rarefaction curve plateaued when the sequence number exceeded 6,000, and the OTU number did not change significantly with an increasing number of sequences thereafter. A minimum of 50,657 sequences were obtained from the tested samples, indicating that the sequence number was sufficient for analysis . PCA basP < 0.05), including Planococcaceae, which was 3.20-fold higher in the healthy group. Compared with healthy rabbits, five genera were significantly reduced in rabbits with diarrhea (P < 0.05), including Clostridium (2.38-fold lower), Roseburia (2.62-fold lower), and Alistipes (3.52-fold lower). At the species level, two species varied significantly between the healthy and diarrhea groups (P < 0.05), including Alistipes indistinctus  and Coprococcus catus (2.89-fold higher in the diarrhea group).Differences in the cecal floras of the diarrhea and healthy groups were analyzed at the phylum, class, order, family, genus, and species levels . At the 3-N and pH increased in the ceca of rabbits with diarrhea. Factors related to the cecal environment, such as pH, VFA, and NH3-N, are known to be closely related to dynamic homeostasis (3-N suggests an increased number of pathogens adapted to the alkaline environment (Weaning induces psychological, environmental, and nutritional stress reactions in rabbits, which leads to diarrhea, dystrophia, and slow growth. In addition, the development of the digestive tracts of young rabbits is a complex and imperfect process. In the present study, we found that diarrhea in young rabbits was most prevalent at 30 days after weaning and caused intestinal dysfunction and changes in the internal environment. The intestinal microbial flora is known to be closely related to host health status. Drastic changes in microbial species and diversity in the intestinal mucosa may cause microbial imbalance and pathogenesis. Our results showed that weaned rabbits with diarrhea had shorter duodenal villi and a lower villus/crypt ratio, implying that diarrhea impaired intestinal development in weaned rabbits. A comparison of the cecal environment between healthy rabbits and those with diarrhea showed that the concentration of acetic acid decreased while NHeostasis . The groeostasis . The incironment .N-acetylmuramoyl-l-alanine amidase, and it can inhibit bacterial growth by degrading peptidoglycan (α, and IL-6 levels in young rabbits.Secreted PGRPs are known to exhibit antibacterial activities . They dodoglycan , whereasdoglycan . In thisRoseburia, Alistipes, and Clostridium. Roseburia is a saccharolytic, butyrate-producing bacterium isolated from the cecum, and includes the species Roseburia hominis. The fecal microbiota of patients with inflammatory bowel disease contains a lower abundance of R. hominis than that of controls (R. hominis colonization was recently demonstrated in the guts of ulcerative colitis patients (Roseburia could be useful for the control and treatment of gut inflammation in rabbit production as well. According to published reports, the genus Clostridium contains several species with different functions. Some of these can produce exotoxins, including Clostridium difficile and Clostridium botulinum (Clostridium butyricum promotes growth performance and immune function and improves the balance of the intestinal microflora in broiler chickens (Clostridium caused diarrhea, but the specific subtype involved was not clear and further experimental studies are required to confirm this finding. In addition, Alistipes was found to differ between healthy rabbits and those with diarrhea. Previous studies have shown that a greater frequency of abdominal pain is correlated with an increased abundance of several bacterial taxa from the genus Alistipes (Alistipes may be associated with abdominal pain in rabbits with diarrhea.Furthermore, the intestinal floras of rabbits with diarrhea and healthy rabbits were analyzed by 16S rRNA V4 amplicon pyrosequencing. The highly conserved region was PCR-amplified using common primers, and the highly variable regions were sequenced for the identification of bacterial species. This technique is an important tool for characterizing microbial floras in environmental samples . The rescontrols . A signipatients . It has otulinum . Howeverchickens . We founlistipes . AlistipAccording to the findings of this study, weaned rabbits are prone to stress reactions, which cause diarrhea and inhibit growth. There are several underlying causes of this, including immune deficiency caused by weaning, an imbalance of the intestinal microflora, and other deleterious factors. Therefore, dietary supplementation of bacteria should be used to improve the composition of the intestinal flora of weaned rabbits. In addition, early feeding, slow weaning, and increased farm sanitation are recommended.Clostridium, Roseburia, and Alistipes between healthy rabbits and those with diarrhea, implying disruption of the microbiotas in the intestines of rabbits with diarrhea. Our findings provide evidence for an association between the intestinal microbial environment and host health in rabbits and contribute to a better understanding of the pathogenesis of diarrhea in young rabbits.In conclusion, diarrhea severely impairs intestinal development and immune function in young rabbits and induces the upregulation of intestinal antibacterial proteins. In addition, pyrosequencing of 16S rRNA V4 in the cecum indicated significant differences in the prevalences of