There are numerous congenital and acquired causes of thrombocytopenia. Thrombocytopenia could be a result of decreased bone marrow production, increased consumption, increased destruction, splenic sequestration or a combination of these causes. In this review, we have focused on some of the serious acquired causes of thrombocytopenia. There have been some significant advances in our understanding of the pathophysiology, diagnostic testing, and treatment of immune thrombocytopenia, heparin-induced thrombocytopenia, thrombotic thrombocytopenic purpura, and atypical hemolytic uremic syndrome over the past five years. These advances have resulted in a significant decrease in mortality and morbidity of patients with these disorders. Despite these advances, we are still faced with numerous unanswered questions in the pathophysiology and management of these complex thrombocytopenic disorders. Acquired thrombocytopenia could be immune or non-immune. Thrombocytopenia could be a result of decreased marrow production, increased destruction or sequestration/consumption in the periphery, or a combination of decreased production and sequestration. Initial steps in the evaluation of thrombocytopenia include review of the peripheral blood smear to exclude pseudo-thrombocytopenia due to platelet clumping. The peripheral blood smear may also provide clues toward other causes of thrombocytopenia when combined with the complete blood count and a good patient history and physical examination. Platelet size and presence of schistocytes, polychromasia, or spherocytes are some of the other features on the peripheral blood smear that help in diagnosing the etiology of thrombocytopenia. Thrombocytopenia could be a harbinger of a serious underlying medical condition such as thrombotic thrombocytopenic purpura (TTP) or heparin-induced thrombocytopenia (HIT). Therefore, it is always important for the treating physician to evaluate thrombocytopenia in a timely fashion so that the treatment for some of the serious conditions is not delayed. The current review will focus primarily on the recent updates in the pathophysiology and management of some of the serious acquired causes of thrombocytopenia encountered in the hospital setting  or steroids, in addition to being therapeutic, might also aid in the diagnosis of ITP. Platelet autoantibodies or T cell-mediated autoimmunity or both play a role in the pathophysiology of ITP resulting in platelet destruction and reduced platelet production2. Although platelet autoantibodies are not used in the diagnosis of ITP, recent work suggests that the lack of platelet-bound antibodies might predict non-responsiveness to rituximab therapy in patients with ITP3. The bleeding phenotype varies significantly in patients with similar platelet counts and may be related to factors such as concomitant medications, age of the patient, and platelet reactivity. There is some evidence to suggest that ITP patients with lower platelet counts and increased platelet reactivity have a lower risk of bleeding4. Intracranial hemorrhage is the most dreaded complication of ITP, and history of significant bleeding from other sites predicts for intracranial hemorrhage5.Most patients presenting with immune thrombocytopenia (ITP) either are asymptomatic or have minor muco-cutaneous bleeding. ITP remains a diagnosis of exclusion because there is no reliable diagnostic test to detect platelet autoantibodies1. High-dose dexamethasone was found in one study to be more effective in inducing long-term remission compared with prednisone6. Multiple cycles of pulsed dexamethasone may result in higher long-term remission rates compared with prednisone or a single cycle of dexamethasone7. Two other retrospective studies showed conflicting results when comparing the efficacy of dexamethasone with that of prednisone9. Further prospective studies are needed to conclusively prove the efficacy of one steroid over the other. Given the convenience of a short-course, high-dose dexamethasone regimen over a longer duration of prednisone, we recommend high-dose dexamethasone over prednisone for the initial treatment of ITP10. Treating physicians should be aware of the fact that a sustained response with high-dose dexamethasone might require multiple cycles of therapy, although some proportion of the patients might not achieve remission despite this approach. IVIG administered at a dose of 1 g/kg for one or two days is the other first-line treatment option1. Given the faster platelet response to IVIG compared with steroids, IVIG is used in conjunction with steroids in patients with severe thrombocytopenia requiring urgent procedures or presenting with moderate to severe bleeding.Steroids are the mainstay in the first-line treatment of ITP. A two- to four-week course of prednisone (1 mg/kg and taper) or four days of pulsed high-dose (40 mg daily) dexamethasone given every two to four weeks are the two commonly used steroid regimens11. There was an increase in the venous thromboembolic events and serious infections resulting in hospitalization in the splenectomized population without a decrease in the overall survival. A recent population-matched cohort study demonstrated that splenectomized patients had a 50% overall increased five-year stroke risk compared with the disease-matched cohort (absolute risk 3% versus 2.3%) but that there was no increase in the five-year stroke risk in ITP patients undergoing splenectomy12. Another recent large single-center study confirmed the curative nature of splenectomy in more than 50% of the patients undergoing the procedure, and corticosteroid dependence predicted for sustained response after splenectomy13. Taking into consideration the high cure rates and the opportunity to avoid long-term medications, we recommend considering splenectomy as the initial second-line treatment option in young steroid-dependent ITP patients.Before the advent of thrombopoietin receptor agonists (TPO-RAs), splenectomy and rituximab were the commonly used second-line agents. A recent single-center cohort study compared 83 splenectomized ITP patients with 83 non-splenectomized ITP patients. Splenectomy resulted in an overall response rate of 52% at 192 months14. A recent randomized control trial comparing rituximab with placebo demonstrated no difference in the complete remission rates between the two arms at 18 months15. Rituximab, when combined with one to three cycles of high-dose dexamethasone in a first-line setting, could result in higher response rates than dexamethasone alone18.Rituximab, an anti-CD20 monoclonal antibody, can result in 50 to 60% initial response rates, although only about 20% of the patients maintain the response at five years21. TPO-RA could also be used in the first-line setting, in conjunction with steroids and IVIG, to treat severe thrombocytopenia and significant bleeding. There is small proportion of patients who are refractory to one of the agents but may respond to the other23. There is also some experience in combining the two agents, given their different target sites on the thrombopoietin receptor, in patients refractory to single-agent, second-line therapy23. TPO-RA could also be used in combination with other immunosuppressive therapies to improve the response rates in refractory ITP patients24.Two TPO-RAs, romiplostim and eltrombopag, were initially used in the third-line setting but have rapidly moved up the ladder to second-line status. These agents have transformed the management for ITP. Long-term follow-up of these agents has demonstrated that they are highly effective and safe in the treatment of patients with ITP25. Unlike the previously studied pegylated human recombinant megakaryocytic growth and development factor (PEG-rHuMGDF), which resulted in the formation of anti-thrombopoietin antibodies27, the new recombinant and glycosylated form of thrombopoietin was similar to endogenous thrombopoietin, and anti-thrombopoietin antibodies were not detected in the limited sample size of patients. These pregnant females with ITP were refractory to the first-line therapy of steroids or IVIG (or both) with some signs and symptoms of bleeding and platelet counts of less than 30 × 109/L. About 75% of patients responded with platelet counts increasing to 30–100 × 109/L or more than 100 × 109/L. TPO-RA and recombinant thrombopoietin might have an important role in the management of pregnant refractory ITP patients.Although ITP in pregnancy can generally be treated with steroids and IVIG, there is an occasional need for other effective therapies when the patients do not respond to the first-line agents. A recent study showed that recombinant thrombopoietin was safe and efficacious in increasing the platelet count in 31 pregnant women with ITP28. HIT is a disease of multiple paradoxes. It is a disease in which an anticoagulant causes thrombosis and thrombocytopenia is associated with thrombosis rather than bleeding. In HIT, unlike in other thrombocytopenias, platelet transfusions increase the risk of clotting. Simply stopping the offending agent (heparin) is not adequate treatment unlike other drug-induced thrombocytopenias and vitamin K antagonists anticoagulant is contraindicated in the treatment of acute HIT.Despite the widespread use of direct oral anticoagulants (DOACs) and other parenteral anticoagulants, unfractionated heparin (UFH) still plays an important role in the management of hospitalized patients. Hence, understanding the pathophysiology and management of HIT is still very relevant to the treating physician29. There are multiple prerequisites for an immunogenic HIT antigen. The immunogenicity depends on distinct stoichiometric ratios of PF4-heparin complexes, and the complexes reach a size of more than 670 kDa by charge neutralization. Immunogenic polyanions with anti-parallel beta-sheet content of more than 30% and an energy release of more than 4,000 calories per molPF4 for effective antibody binding are the other prerequisites31. The atomic structure of the PF4-heparin complex was worked out in 2015, although the synthetic pentasaccharide, fondaparinux, was used in the complex instead of heparin32. PF4 released from platelets during a bacterial infection can bind to the bacterial cell wall, resulting in a priming step that could result in the formation of antibodies against PF4-heparin complex when exposed to heparin at a later time33. PF4-heparin complexes can activate complement and bind to the complement receptor 2 (CD21) on B cells, enhancing the antibody production34. A better understanding of the HIT pathophysiology would lead to the development of better diagnostic tests and effective anticoagulants to treat the condition.HIT antibodies are induced by platelet factor 4 (PF4)-heparin or endogenous glycosaminoglycan complexes. A subset of these IgG antibodies can cross-link Fcγ receptor IIA (FcRγIIA), causing platelet and monocyte activation that results in consumptive thrombocytopenia and venous and/or arterial thrombosis35. The tissue damage as a result of total hip or knee arthroplasty can lead to platelet activation, polyanion release, and formation of PF4-polyanion complexes. Dynamic mechanical thromboprophylaxis might add to the tissue damage, platelet activation, and polyanion release, increasing the incidence of HIT antibody formation36. In the setting of total knee arthroplasty, the incidence of HIT antibody formation was similar in patients receiving dynamic mechanical thromboprophylaxis with or without heparin/fondaparinux. Since they were identified by enzyme-linked immunosorbent assay (ELISA), it is not clear whether these antibodies are platelet-activating.Spontaneous HIT develops in the absence of exposure to any heparin and is common in patients undergoing orthopedic procedures37. Although the pharmacy-and-therapeutics committee of many hospitals prefers UFH over LMWH for venous thromboembolism prophylaxis because of the cost differential between the two drugs, a single case of complicated HIT with thrombosis can wipe out all the upfront cost savings.Reducing the incidence of HIT with the use of low-molecular-weight heparins (LMWHs) instead of UFH results in significant cost savings with an ‘avoid heparin’ protocol39. The time taken to achieve therapeutic levels, less risk of bleeding, and shorter hospital stay resulting in cost savings are some of the advantages of fondaparinux over argatroban. We recommend fondaparinux over parenteral direct thrombin inhibitors in the treatment of acute HIT in patients who are not critically ill and have good renal function. There is accumulating evidence for the safety and efficacy of DOACs in the treatment of HIT41. These medications, like fondaparinux, would definitely help with reducing the length of hospital stay and patient convenience. Until there are more data, we do not recommend DOACs in the initial treatment of acute HIT, but they could be used in patients after platelet recovery.Fondaparinux is safe and effective in the treatment of acute HIT42.A small proportion of patients with HIT might have refractory disease with prolonged thrombocytopenia despite therapeutic anticoagulation. IVIG could be used in conjunction with anticoagulation to help with platelet recovery43. TTP could be acquired due to autoantibodies to ADAMTS13 or congenital due to severe deficiency of ADAMTS13. We recommend testing for ADAMTS13 in every patient with MAHA and thrombocytopenia when TTP is part of the differential diagnosis, although treatment of TTP should not be delayed while awaiting the results44. Sepsis and cirrhosis can decrease ADAMTS13 activity but generally not less than 10%43. Hyperbilirubinemia (>10 mg/dL) and free plasma hemoglobin, however, could give falsely deficient levels (<10%) due to interference in the fluorescence resonance energy transfer (FRET) assay45. IgG antibodies against ADAMTS13 are detected in 75–80% of the patients by the functional Bethesda method in acquired TTP; however, remaining patients may have non-neutralizing antibodies often detected by an ELISA46. Because ADAMTS13 assay is often sent to a reference laboratory, most patients with unexplained MAHA and thrombocytopenia are started on plasma exchange (PLEX). If the ADMTS13 is more than 10%, PLEX should be discontinued and other causes of MAHA and thrombocytopenia should be investigated for appropriate treatment such as metastatic malignancy, malignant hypertension, and atypical hemolytic uremic syndrome (aHUS)44. The blood for ADAMTS13 testing should preferably be drawn prior to instituting PLEX, although levels are often low in acquired TTP even after a couple of days of PLEX47. Not achieving ADAMTS13 activity levels of more than 10% within seven days of instituting daily PLEX could be associated with worse outcomes47.Microangiopathic hemolytic anemia (MAHA) and thrombocytopenia are a common denominator in multiple disease states. Of these, TTP is the easiest to diagnose since it is caused by a severe ADAMTS13  deficiency (<10%)48. Glucocorticoids are often started simultaneously. Once the diagnosis is confirmed to be autoimmune TTP, rituximab is routinely used in the treatment of relapsed or refractory TTP patients. However, there is evidence to support upfront use of rituximab in acute TTP to reduce exacerbation, refractoriness, number of PLEX sessions, and possibly hospital stay46. Although this approach might result in overtreatment of half of the patients with TTP, the decreased morbidity and mortality in patients with refractory or relapsed TTP would outweigh the overtreatment issue. Most physicians use rituximab 375 mg/m2 weekly for four doses, although our personal experience with a lower dose of rituximab at 100 mg weekly for four doses appears promising. There is a clinical trial under way to study the efficacy of low-dose rituximab . Bortezomib is very promising as an adjunct to PLEX in the treatment of refractory or relapsed acquired TTP or both50. We recommend using bortezomib in refractory and/or relapsed TTP patients who do not respond to rituximab. Daily subcutaneous caplacizumab, a nanobody against von Willebrand factor (VWF) A-1 domain preventing its interaction with GPIb on platelets, resulted in faster platelet responses in acute TTP compared with a placebo when used along with PLEX51. This nanobody is not a cure; however, by preventing the interaction between VWF and platelets, it causes rapid reversal of the underlying pathogenetic mechanism, resulting in a potential to reduce early mortality. The drug is being studied further in a phase 3 trial.PLEX should be started as soon as the clinical diagnosis of TTP is made. The currently available plasma preparations are all equivalent in terms of their outcomes in TTP52. Although measuring ADAMTS13 activity during remission and preemptively treating with rituximab for moderately to severely low ADAMTS13 activity is a commonly used approach at some institutions, this practice needs further evaluation. We recommend following these patients carefully because of the higher rate of TTP recurrence in this population. Since this is a very episodic disease, education of patients for signs and symptoms of relapse is a crucial part of the management plan. In addition to the neurological and renal manifestations, TTP could result in cardiac and mesenteric ischemia54. Patients could commonly have elevated cardiac troponin without any clinical cardiac abnormalities53. Similarly, long-term neurocognitive defects are recognized in patients with TTP56.Some patients achieving clinical remission may have ADAMTS13 activity of less than 10%. Nonetheless, ADAMTS13 activity of less than 10% alone is not sufficient for TTP relapse and requires a trigger such as infection, pregnancy, or concomitant inflammatory conditions increasing VWF levels57. Although MAHA, thrombocytopenia, and oliguric renal failure are the three common presenting features, aHUS is a systemic disease that can involve any organ system. aHUS, unlike Shiga toxin-producingEscherichia coli HUS, does not present with the prodrome of bloody diarrhea and often is recurrent. Mutations in the genes encoding for complement proteins, complement regulatory factors, or autoantibodies to complement factors, when combined with another stressor such as infection or surgery, often result in the development of aHUS. Only about 60% of patients with aHUS have an identifiable genetic mutation58. Mutations in the gene encoding for factor H are the most common among patients with aHUS. The other defects involve mutations in CD46, factor I,C3, factor B, thrombomodulin, diacylglycerol kinase ε, and CFHR1/3 deficiency with anti-factor H autoantibodies. Since mutation testing and screening for antibodies to complement proteins are not available readily, diagnosis of aHUS is usually made on the basis of clinical features and an ADAMTS13 activity of more than 10%. A modified Ham test has shown some promise in distinguishing aHUS and TTP serum samples59. Genetic mutations or the autoantibodies in aHUS result in the activation of alternate pathway (AP); whenPIGA null TF-1 cells (PNH-like cells) are added to the serum samples, aHUS serum samples result in a positive test (cell death) because of the activated AP compared with the TTP samples.aHUS results from a dysregulated complement system, leading to cell and tissue damage60. The optimal duration of eculizumab therapy in aHUS is not clearly defined at the present time61.If TTP is in the differential diagnosis, PLEX is started until ADAMTS13 activity results. But if the presenting features are classic for aHUS or if the ADAMTS13 activity is more than 10%, the appropriate management would be to start eculizumab, an anti-C5 antibody, immediately. Eculizumab is effective in improving renal function and platelet count and halting tissue damage from the AP activationIn summary, acute thrombocytopenia is a common clinical presenting feature in many serious hematological conditions. There have been some significant advances in our understanding of the pathophysiology, diagnostic testing, and treatment of these disorders over the past five years . Unfortu


Analogous to DNA methylation and histone modifications, RNA modifications represent a novel layer of regulation of gene expression. The dynamic nature and increasing number of RNA modifications offer new possibilities to rapidly alter gene expression upon specific environmental changes. Recent lines of evidence indicate that modified RNA molecules and associated complexes regulating and “reading” RNA modifications play key roles in the nervous system of several organisms, controlling both, its development and function. Mutations in several human genes that modify transfer RNA (tRNA) have been linked to neurological disorders, in particular to intellectual disability. Loss of RNA modifications alters the stability of tRNA, resulting in reduced translation efficiency and generation of tRNA fragments, which can interfere with neuronal functions. Modifications present on messenger RNAs (mRNAs) also play important roles during brain development. They contribute to neuronal growth and regeneration as well as to the local regulation of synaptic functions. Hence, potential combinatorial effects of RNA modifications on different classes of RNA may represent a novel code to dynamically fine tune gene expression during brain function. Here we discuss the recent findings demonstrating the impact of modified RNAs on neuronal processes and disorders. An estimated 1–2% of all genes in a given organism contribute to nucleic acid modification systems, suggesting biological importance of modified nucleotides , pseudouridine (Ψ) and ribose 2′-O-methylation (Nm) on mRNA  and 5-methylcytidine (m5C) is still debated   Figure . VariousAsp, tRNAVal, and tRNAGly, has been described in yeast, Drosophila, mouse, and human cells  differentiation and motility  is a common nucleoside modification of RNA, where a methyl group is added to the 2′ hydroxyl of the ribose moiety Figure . Nm incrFTSJ1 gene (OMIM:300499)  and mutations in the Ser . Two families of snoRNAs have been described, based on their structure and function: C/D box snoRNAs are responsible for 2′-O-methylation , memory default, and shorter lifespan , are viable but exhibit memory impairment and shortened lifespan  from 3′ → 5′ exonuclease degradation  particule catalyzes the uridine modification  is the first discovered     and ALKBH5 , which strongly impaired neuronal differentiation  are m6A modified in human, but not in mouse cultures of neuronal progenitor cells (NPC), raising the possibility that m6A regulates specifically these human diseases  were elevated, which led to increased translation during the time of axon regeneration, via the specific m6A reader protein Ythdf1. Mettl14 and Ythdf1 conditional KO mice displayed strong reduction of sensory axon regeneration, resulting from reduced protein synthesis, revealing the critical role of m6A modification in response to injury , was recently shown to control axon outgrowth, branching and to regulate synaptic bouton formation via the activity of the CCAP/bursicon neurons , to repress translation of a subset of FMRP target mRNAs  and AlkB homolog 5 (ALKBH5) , suggesting an involvement of ALKBH5 in conferring risk of MDD  that may carry distinct functions (Briggs and Dinman, 6A clearly demonstrate the involvement of mRNA modification in different aspects of neuronal development and regulation. The large diversity of RNA processing events in the brain, including the high rates of alternative splicing and recursive splicing (Duff et al., 6A modification was recently found on circRNAs, and enables their translation (Yang et al., 6A brain functions may rely on circRNA-mediated regulation. Thus, because neurons face distinct challenges with regards to localisation of RNAs to distal processes and to localized translation it is likely that in the brain, more than in any other tissues, a combinatorial effect of RNA modifications on different classes of RNAs represents a critical informational layer that dynamically fine-tunes gene regulation (Figure That being said, the general picture is probably more complex. For instance, recent reports show that tRFs not only interfere with translation but can also affect transposon regulation and genome stability (Durdevic et al., n Figure . ExcitinAll 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.


Geobacillus stearothermophillus ATCC-7953 was used, with a microbialpopulation of 106UFC/Filter paper substrate, removed from the biologicalindicator. Three of them were introduced into each instrument at the time ofassembly, and sterilized at pressurized saturated steam, 134oC for 5 minutes.After sterilization, the instrument was disassembled and each filter papersubstrate was inoculated in soybean casein culture and incubated at 56oC for 21days. In case of absence of growth, they were subjected to heat shock of 80oC, for20 minutes and re-incubated for 72 hours. Sample size: 185 graspers and 185trocars, with 95% power. We paired the experiments with comparative negativecontrol groups  and positive control ,subject to the same incubation procedures. there was no microbial growth in experimental and negative control. The results ofthe positive control were satisfactory. this study provided strong scientific evidence to support the safety of steamsterilizing of the assembled laparoscopic instrument. The videolaparoscopic surgery is a technological innovation that has emerged as analternative to surgical, diagnostic and therapeutic procedures, which were usuallyperformed through laparotomy. This technique has indisputable advantages for thepatients, and new challenges for the nurses responsible for the Sterile Supply Center(SSC), including the establishment of guidelines for the safe processing of instrumentsand accessories with complex conformation, understood as those with less than 5 mm oflumen or blind-end, inaccessible internal spaces for direct friction, holes orvalvesOn the issue of sterilization, the pressurized saturated steam is the preferred methodfor heat resistant laparoscopic instrumental because it brings advantages such as lowD,-,The classical recommendations state that the heat resistant surgical instruments are tobe open, disassembled and with the surfaces free for steam sterilizationAmong health professionals, there is a deep-rooted concept that to achieve the successof the sterilization by the saturated pressure steam autoclave, direct contact of steamwith all surfaces of materials is necessary, without considering the physical principlesof latent heat. There is a need to question rooted concepts based on traditions, andstrong scientific evidence should be sought in order to support decision-making inhealthcare practice.As laparoscopic accessories are complex instruments with several pieces of small size,if sterilized when completely dismantled, they can present problems for the surgicalteams at the time of assembly in the operative field. It is noteworthy that somesurgical scrub aides are unaware of the correct assembly, compromising theirfunctionality, creating stress and disrupting the start of the surgical procedure.The autoclaving of preassembled laparoscopic instruments is a reality identified by asurvey, with a sample of 263 nursing professionals, in which 37% of respondents reportedthat in their institutions they sterilized the assembled laparoscopic instruments--The scientific literature does not provide a conclusive answer about the safety ofsaturated steam pressure sterilization, of the assembled laparoscopic instrument Given the abovementioned result, this research aimed to evaluate the safety of steamsterilization, of the assembled laparoscopic instrument with challenge infection, inorder to bring to the table robust scientific evidence to support the decisions of thenurses that manage the SSC, focusing on the safety of the surgical patients.This study is characterized as laboratory-based experimental. As test specimen weselected two types of reusable laparoscopic instruments that are more complex for reuse:Trocar with screw windowed valve, made of five parts, being one of them with a lumen of5 mm diameter and 5 mm Dissection Forceps made of four parts with teethed end, lumen of30 cm long and internal diameter of 3mm. The laparoscopic instruments used in theresearch were specific for this purpose and were not previously used in humans.Geobacillusstearothermophillus ATCC-7953 in sporulated form, a biologic indicatoravailable in the market to monitor steam pressurized sterilization cycles(AttestTM(r) biologic indicator, reference 1.262, readings after 48 hs,steam). The self-contained biologic indicator is built in a paper substrate (2.5X0.5 cm)with a minimum of 100,000 calibrated dry spores of Geobacillusstearothermophillus ATCC-7953. We chose this microorganism because it is thestandard for biologic monitoring of the efficacy control in autoclave cycles, due to itsresistance to humid heat and low pathogenic conditions under normal conditionsThe selected challenge microorganism was the oC for 48 hours. Three groups were defined: an experiment group, a negative control and a positivecontrol. In the experimental group we analyzed the results of microbial culture from 370assembled laparoscopic instruments, coming from 185 graspers and 185 trocars, for atotal of 1080 sampling units. This sampling size showed to have a 95% sample power, inwhich the chance of the assembled instrument of presenting viable spores aftersterilization is at least 8%. As negative control group, 10 disassembled laparoscopicinstruments were analyzed, composed of 5 graspers and 5 trocars, for a total of 30sampling culture units. The positive control was a set of 30 non-sterilized paper filtersubstrate, inoculated seeding directly in TSB, 56Geobacillus stearothermophillus ATCC-7953were separated. Three units of paper substrate were placed inside of each laparoscopicinstrument in the process of assemblage  model 6412HF,558 liters, micro processed, thermal qualification for sterilization of surgicalmaterial at 134 Co for 5 minutes.The instruments were packed individually in surgical grade paper and autoclaved inpressured saturated steam, with pre-vacuum autoclave CisaTryptic Soy Broth (TSB) culture medium, incubated at56o C for 21 days. If no microbiologic growth was observed after thistime, tubes were exposed to a thermal shock during 20 minutes at 80o C,re-incubating for 72 hs. at 56oC for a final readingAfter sterilization, the instruments were disassembled inside the biologically protectedcabinet using aseptic techniques, and each biologic indicator paper substrates wasseeded in o C during 7 daysThe TSB culture medium that were used in the experiment were prepared form dehydratedmedia, as per manufacturer recommendationThe results of the experiments are presented in oC for 5 minutes,Geobacillusstearothermophillus ATCC-7953 in three times 106 UFCconcentration, sterilization tests with direct inoculation method and a sample size thatdemonstrated robust results.The present laboratory controlled research succeeded in sterilizing assembledlaparoscopic instruments in pressurized saturated steam, thus proving the microbiologicsafety of this procedure. An thermally qualified autoclave was used following therecommended parameters for pressurized saturated stem with pre-vacuum, at134,-Autoclave sterilization of assembled laparoscopic instruments is a reality in Brazilianhealthcare facilities-Sterilization of assembled laparoscopic instruments was studied previouslySerratia marcescens) and sporulated bacteria(Bacillus subtilis e Bacillus stearothermophilus) as challengecontamination of two laparoscopic graspers and two trocars (5mm and 10mm respectively).The inoculation technique and retrieval was done through swabs, retrieving the challengemicroorganism both in assembled and disassembled laparoscopic sterilized instruments. Inspite of the fact that the swab technique allows for a quantitative evaluation, it haslimitations in standardizing the rolling resistance, the angle and the pressure degreeduring the procedure, it is not able to control reproducibility, and results have alarge degree of variabilityThe first researchGeobacillus stearothermophilus in far higher concentration than theconcentration found in worst case scenarios in clinical practice, associated with directinoculation sterilization tests, ensuring full retrieval of viable microorganisms,respecting the incubation timing to allow the possible surviving spores may germinateafter the thermal shock physical stimulus.In this same researcho C inconventional and flash cycles with exposures of 10 and 3 minutes respectively. Allvegetative microorganisms were eliminated with conventional and flash cycles ofsterilization. Filling of the lumen with organic material showed resistance to directsteam contact, similar to the case when the laparoscopic instruments are sterilizedwhile assembled.A different researchGeobacillusstearothermophilus ATCC 7953 in the trocar lumen without hamburger meat anddifferent time exposures, 3, 4,5 and 6 minutes. Only when time exposure was extendedfrom 7 to 10 minutes the spores were fully destroyed. These results are in favor of thelatent heat microbial destruction, in spite of the hard scenario of challengecontamination and massive organic material.In the same conditions, with organic material as lumen filling o C in 4 minutes,Geobacillus stearothermophilus ATCC 7953 using pressurizedsaturated steam with pre-vacuum sterilization cycle at 134 o C in 5minutes.As the standard parameters for pressurized saturated steam with pre-vacuum autoclave are134Geobacillusstearothermophilus ATCC 7953 with 10% of lamb blood. Instruments passedthough automated cleaning in ultrasonic washer with intermittent flow and hand cleaningbefore assembling and sterilization under pressurized saturated steam with pre-vacuum at134134 o C in 5 minutes. No microorganisms were retrieved in this group,reinforcing the chance of safety of sterilization under pressurized saturated steam ofassembled instruments.Another researchGeobacillus stearothermophilus 106 UFC were placed inside each laparoscopic instrument before sterilization, thuscreating a three fold 106 UFC challenge of the test microorganism in eachsample unit.ResearchersSterilization under pressurized saturated steam of assembled laparoscopic instruments ismicrobiologically safe, breaking with the paradigm of classic recommendations ofautoclaving only disassembled material. Results of this research, under the experimentconditions, are strong scientific evidence that supports a systematic review of thistopic and gives inputs to the decision-making process related to the microbiologicalsafety of pressurized saturated steam sterilization of assembled laparoscopicinstruments. Additionally, it is desirable that it may give inputs to lawmakers toformalize the possibility of autoclaving pre-assembled laparoscopic instruments.


Homeobox genes are master regulators of cell fate during embryonic development and their expression is altered in cancer. By regulating the balance between cell proliferation and differentiation, they maintain homeostasis of normal tissues. Here, we screened the expression of homeobox genes in mammary stem cells to establish their role in stem cells transformation in breast cancer.HOXC8 epigenetic regulation was studied by DNA methylation and miRNA expression analyses. Self-renewal and differentiation of HOXC8-overexpressing or knockdown cells were assessed by flow cytometry and mammosphere, 3D matrigel and soft agar assays. Clinical relevance of in vitro findings were validated by bioinformatics analysis of patient datasets from TCGA and METABRIC studies.Using a Homeobox Genes PCR array, we screened 83 homeobox genes in normal cancer breast stem/progenitor cells isolated by flow cytometry. The candidate gene HOXC8 was consistently downregulated in stem/progenitor cells of all breast molecular subtypes, thus representing an interesting tumour suppressor candidate. We show that downregulated expression of HOXC8 is associated with DNA methylation at the gene promoter and expression of miR196 family members. Functional studies demonstrated that HOXC8 gain of function induces a decrease in the CD44+/CD24-/low cancer stem cell population and proportion of chemoresistant cells, with a concomitant increase in CD24+ differentiated cells. Increased HOXC8 levels also decrease the ability of cancer cells to form mammospheres and to grow in anchorage-independent conditions. Furthermore, loss of HOXC8 in non-tumorigenic mammary epithelial cells expands the cancer stem/progenitor cells pool, increases stem cell self-renewal, prevents differentiation induced by retinoic acid and induces a transformed phenotype.In this study we demonstrate altered expression of homeobox genes in breast cancer stem/progenitor cells. Taken together, our study points to an important role of homeobox genes in breast cancer stem/progenitor cell function and establishes HOXC8 as a suppressor of stemness and transformation in the mammary gland lineage.The online version of this article (doi:10.1186/s12943-017-0605-z) contains supplementary material, which is available to authorized users. Cancer stem/progenitor cell cells (CSC) are considered to play an important role in breast cancer complexity as they retain the fundamental features of normal stem cells, being able to self-renew and differentiate into the different cell types that comprise the heterogeneous tumour mass , 2. BreaGenes that are required for tissue development and normal stem cell plasticity may contribute to the molecular blueprint of breast CSC and tumorigenesis. Indeed, it is well established that many molecular pathways involved in normal development are altered during cancer progression . AmongstHoxc6 and Msx2 being down-regulated and up-regulated by estrogen and progesterone, respectively. Msx2 is also regulated by the interaction between epithelial and stromal cells in the gland , wi, wiHOX cnescence . These fnescence  become pnescence , 45. Ournescence , 47. DNA outcome . Express outcome . TherefoHOXC8 was found consistently downregulated in CSC and therefore represented a novel putative tumour suppressor candidate. HOXC8 was especially downregulated in TN and HER2-E CSC and it was also found to be downregulated in clinical samples of TCGA and METABRIC datasets, strengthening its significance as a potential biomarker. In line with previous studies reporting the importance of epigenetic regulation of homeobox genes during development and carcinogenesis [HOXC8 silencing is associated with the expression of miR196 and DNA methylation at the gene promoter region in CSC. Regulation of HOXC8 via miRNA 196 has been reported in breast cancer whereby the ration of miR196 and HOXC8 correlates with cell migration and metastasis [HOXC8 promoter can induce gene silencing by inducing polymerase II stalling during transcription [Among the differentially expressed genes identified in this study, ogenesis , we alsotastasis  and methcription .HOXC8 confers mammary cells with stemness potential and this can be reversed by its re-expression with induction of differentiation. The enhanced CSC population induced by downregulation of HOXC8 in mammary cells is also associated with an increase in transformed phenotype and this observation supports the notion that breast cell tumorigenicity is enhanced through acquisition of an undifferentiated state [Our results demonstrate that HOXC8 is an important regulator of stem cell self-renewal and that HOXC8 can act as modulator of cell differentiation in the breast. Using a combination of strategies and cell culture models, we show that silencing of ed state . HOX proed state , 16, 17.ed state , 53.HOXC8 is expressed in the neural tube and somatic mesoderm and its expression is essential for skeletal development [HOXC8 is also expressed in the normal mammary gland [HOXC8 has been reported to act either as tumour suppressor gene or oncogene, suggesting that the function of HOXC8 may depend on the activation or repression of different HOXC8 targets whose expression can be tissue-specific. Several targets of HOXC8 have been characterised [During development, elopment , 55. HOXry gland , 57 and ry gland . Previoury gland , 59. Thiry gland , 60–63. cterised  and theicterised , targetiHOXC8 can be induced by retinoids in normal breast cells. This role has also been recently demonstrated for HOXA5, suggesting a prominent role of HOX genes in the differentiation of the mammary gland [We have also shown that HOXC8 downregulation in mammary cells can lead to impaired differentiation in response to retinoic acid, and that the expression of ry gland . Retinoiry gland , 67. TheHOXC8 demonstrated to function as a possible novel tumour suppressor in breast CSC by regulating stem cell self-renewal, differentiation and transformation. Therefore, this study demonstrates the critical role of homeobox genes in stem cells and paves the way to larger functional studies to explore the networks involved in homeotic gene regulation in breast cancer.This study reports the screening of a large number of homeobox genes in breast CSC. We show that the expression of homeobox genes is altered in early-transformed breast cells and in different breast cancer molecular subtypes. Altered expression of homeobox genes was also supported by data obtained from large cohorts of patient through bioinformatics analysis. Therefore, this screening provides novel biomarkers that can be used in precision medicine and as possible targets for CSC-directed therapies. Of the altered genes,


Acute Thrombotic Thrombocytopenic Purpura (TTP) is a rare haematology emergency, associated with multi organ microthrombi formation and a high mortality if untreated. Most cases are immune mediated. Congenital TTP, thought to affect 1/million of the population, is associated with inherited homozygous or compound heterozygous defects resulting in an absolute enzyme deficiency. Congenital TTP may present in the neonatal period/childhood or delayed until adulthood, primarily pregnancy.Currently, therapy requires replacement of the missing enzyme, ADAMTS 13 . ADAMTS 13 is a zinc containing metalloproteinase enzyme with a unique relationship in ensuring haemostasis with its substrate VWF. Cleavage of UL VWF (ultra large von Willebrand factor) multimers by ADAMTS 13, results in haemostatically active forms. A deficiency of ADAMTS 13, as in congenital TTP or inhibition due to immune mediated disease, is associated with severely low ADAMTS 13 activity levels (<10%). This results in excess platelet binding to UL VWF multimers and resultant microvascular thrombi.An enzyme deficiency in TTP was only confirmed 20 years ago , 2 and cCompleted, is the first in human, phase 1 study of recombinant ADAMTS 13 protein, developed in CHO cells, confirming its use was safe and efficacious in congenital TTP patients . FifteenWhat does this mean for the future of TTP treatment? Recombinant ADAMTS 13 may avoid the high adverse reaction rates with standard plasma as well as pathogen transfer risks. In congenital TTP, delivery of plasma infusion over a lifetime can be difficult because of repeated reactions. The risk is an inability to adequately treat patients because of significant side effects from therapy and the result is end organ damage affecting vital organs such as the heart, brain or kidneys. Recombinant ADAMTS 13 provides a pure replacement of the missing enzyme. The smaller infusion volume may allow for higher ADAMTS 13 activity levels to be achieved to more optimally treat the disease. Small volumes of this specific enzyme and ease of administration should significantly improve the immediate and longer-term impact of disease.Potential future roles for recombinant ADAMTS 13: Currently a single volume plasma exchange in iTTP involves 3-5 litres of plasma, insertion of a central venous catheter and associated potential complications. Planned studies in iTTP aim to demonstrate the utility of replacement strategies with small volumes of pure ADAMTS 13 protein, with the potential to transform the treatment of this life-threatening condition. High VWF levels (and consumption of ADAMTS 13) are seen in a number of medical conditions, most notably stroke and cardThe development and use of recombinant ADAMTS 13 in TTP is a landmark achievement in haemostasis and thrombosis in this decade. It has the potential to offer a pure, safe therapy for congenital and immune mediated TTP that may revolutionise treatment pathways: once the trials of safety and efficacy are complete. The significant success from the Phase I data will allow progress of these trials and important improvements, ultimately, in patient care.


Listeria monocytogenes into host cells was first proposed in 2002. Since then, work has shown that septins assemble in response to a wide variety of invasive bacterial pathogens, and septin assemblies can have different roles during the bacterial infection process. Here we review the interplay between septins and bacterial pathogens, highlighting septins as a structural determinant of host defense. We also discuss how investigation of septin assembly in response to bacterial infection can yield insight into basic cellular processes including phagocytosis, autophagy, and mitochondrial dynamics.Septins, a unique cytoskeletal component associated with cellular membranes, are increasingly recognized as having important roles in host defense against bacterial infection. A role for septins during invasion of  Work has shown that components of the cytoskeleton occupy a central role in innate immunity by promoting bacterial sensing and executing antibacterial functions Mostowy, . On the Saccharomyces cerevisiae as crucial for cell division , and 13 in both mice and humans (SEPT1-12 and SEPT14). Vertebrate septins can be classified into 4 subgroups, namely the SEPT2, SEPT3, SEPT6, and SEPT7 subgroups, based on homology of sequence and protein domains  and facilitates the host cell entry of invasive bacteria . In this section, we summarize how several bacterial pathogens manipulate septins at the plasma membrane to support bacterial adherence to the host cell and enable bacterial internalization into both immune and non-immune cells.Bacterial adhesion to the host cell is fundamental for some pathogens to establish infection. Adhesion prevents the mechanical clearance of extracellular bacteria  is a Gram-negative bacterium responsible for diarrheal disease in humans. By using a type III secretion system (T3SS), a molecular syringe that secretes effector proteins into the host cell, EPEC injects Translocated Intimin receptor (Tir) into the plasma membrane so that it functions as an anchor for Intimin, an effector protein located on the EPEC outer membrane  and E. coli secretion protein F (EspF) cause abnormal and mislocalized septin assemblies that affect morphogenesis and cell division , a toxin that mediates formation of microtubule-based protrusions, which wrap around bacteria and increase their adherence to the host cell , a protein which associates to the plus end of microtubules, suggesting that septins guide microtubule polymerization in the extending protrusion  and Internalin B (InlB) with plasma membrane proteins E-cadherin and Met, respectively  into the host cell cytosol and increases the size/flexibility of the inclusion to enable bacterial replication  have evolved different mechanisms to polymerize actin tails . Surprisingly, proteomics also revealed that 21.4% of septin cage-associated proteins were annotated as exclusively mitochondrial. Indeed, high-resolution microscopy showed that mitochondria support septin assembly into the cages that entrap Shigella for autophagy. A role for mitochondria in septin cage assembly was confirmed by depletion of factors implicated in mitochondria fission and fusion , a pore-forming toxin . Considering the emerging roles for the host cytoskeleton in cell-autonomous immunity (Mostowy and Shenoy, in vivo (Mostowy et al., in vivo systems, including the zebrafish and mouse, will be essential to illuminate a more complete understanding of septin biology during host defense against bacterial infection.Investigation into the cell biology of bacterial infection has significantly improved our understanding of both infection and cytoskeleton biology (Haglund and Welch, VT and SM jointly wrote the manuscript.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.


A fractional dose of inactivated poliovirus vaccine (fIPV) administered by the intradermal route delivers one fifth of the full vaccine dose administered by the intramuscular route and offers a potential dose-sparing strategy to stretch the limited global IPV supply while further improving population immunity. Multiple studies have assessed immunogenicity of intradermal fIPV compared with the full intramuscular dose and demonstrated encouraging results. Novel intradermal devices, including intradermal adapters and disposable-syringe jet injectors, have also been developed and evaluated as alternatives to traditional Bacillus Calmette–Guérin needles and syringes for the administration of fIPV. Initial experience in India, Pakistan, and Sri Lanka suggests that it is operationally feasible to implement fIPV vaccination on a large scale. Given the available scientific data and operational feasibility shown in early-adopter countries, countries are encouraged to consider introducing a fIPV strategy into their routine immunization and supplementary immunization activities. Providing vaccination through the intradermal route is an effective means of delivering vaccines due to the high concentration of immune cells in the epidermis (outermost layer of skin) . ConsequThe concept of a reduced—or fractional—dose of inactivated poliovirus vaccine (fIPV) through intradermal administration has been evaluated in a number of clinical trials over the past 50 years. A study reported by Jonas Salk in 1953 using the first generation of IPV (before enhanced-potency IPV became available) concluded that polio vaccination given by both the intradermal route (using one sixth to one tenth of the standard dose) and intramuscular route demonstrated substantial immunogenicity . Trials In 2012, the World Health Assembly (WHA) endorsed the proposed Polio Endgame Strategy, which includes withdrawal of the Sabin-virus type 2 antigen—responsible for an estimated 95% of vaccine-derived cases of polio —by replaAt the same time, the WHA requested that the World Health Organization (WHO) work with partners and manufacturers to “enhance the affordability, effectiveness and accessibility [of IPV]” . To fulfThe possibility of using fIPV has recently drawn more attention after the two global suppliers of WHO-prequalified IPV reported significant challenges in scaling up production to meet the new demand, resulting in a >50% reduction in their initial supply commitments made in 2014 (for the period 2014–2018). In many countries, these constraints have resulted in a delay in IPV introduction or stockouts, and they have affected the global IPV reserve established for outbreak response and supplementary immunization campaigns . TherefoFurthermore, even after polio eradication is achieved worldwide, countries will need to maintain population immunity against possible undetected polio transmission and accidental release of poliovirus from laboratories or vaccine production facilities . FollowiIn this context, we provide an overview of (1) the recent scientific literature on the use of fIPV for the primary series  and as a booster dose; (2) the development of new, improved devices for intradermal delivery of vaccines; and (3) the regulatory and operational aspects of using fIPV, including recent field experience.Humoral immunity (antibody response) protects an individual from paralytic disease. A series of clinical studies, many conducted within the past 5 years, have evaluated the humoral immunity of fIPV  administered intradermally through a variety of vaccine delivery devices  compared with a full intramuscular dose when provided as primary vaccination in a 2- or 3-dose series using different vaccination schedules. These studies—conducted in Cuba , 13, OmaThe studies found that cumulative seroconversion rates  for all polio serotypes following the complete vaccination series were comparable between the fIPV and intramuscular IPV groups when there was less interference with maternal antibody . On the other hand, the results were varied when the vaccination series started earlier (such as at 6 weeks of age). The Philippines study showed equivalent immunogenicity between fIPV and intramuscular IPV when each vaccine was administered at 6, 10, and 14 weeks of age . HoweverBecause most OPV-using countries have added a single dose of IPV in their primary vaccination series, it is also useful to compare the immunogenicity of 2 fIPV doses with that of a single full intramuscular dose. In all studies, 2 fIPV doses  resulted in substantially higher seroconversion rates for all poliovirus serotypes than a single intramuscular dose , 16, 17 Examining the median antibody levels by vaccination group in these studies, a single fIPV dose of 0.1 mL resulted in lower median neutralizing antibody titers (MATs) for any of the 3 poliovirus serotypes compared with a single full (0.5 mL) intramuscular dose. However, the MATs in the fIPV groups were all >1:8 dilution, indicating protection against paralytic disease. Thus, the difference in antibody levels between the fIPV and intramuscular groups was not considered clinically relevant . In addiAfter reviewing these data in October 2016, WHO’s Strategic Advisory Group of Experts on Immunization (SAGE) reiterated the recommendation it first made in April 2016 that countries should start preparing for a 2-dose fIPV schedule , in lieu of a single intramuscular dose at 14 weeks . Prior tAlthough humoral immunity is important to prevent paralytic disease, mucosal immunity through the intestinal tract is essential to prevent excretion and transmission of the virus. Some of the studies mentioned above compared intestinal immunity induced by fractional versus full-dose IPV—with inconclusive results.In the Oman study, infants received either a full intramuscular or intradermal fIPV at 2, 4, and 6 months of age, followed by a challenge dose of monovalent OPV type 1 (mOPV1) at 7 months . Seven dA study in the Philippines compared seroprotection of fractional and full-dose IPV 12 to 15 months after infants received the primary series with 1 of these formulations at 6, 10, and 14 weeks [Studies assessing the ability of fIPV to boost immunity among children who had previously received OPV have been conducted in India , Cuba 2, and GamBased on this new evidence, the WHO SAGE recommended in October 2016 that outbreak response campaigns (to boost population immunity) should use only fIPV .One of the challenges with the fIPV strategy is that intradermal injections are more complex to administer than intramuscular injections. Health workers must be well trained in the technique, and incorrect administration can lead to adverse events. However, new injection technologies have recently been developed and registered as alternatives to the conventional BCG needle and syringe to facilitate intradermal delivery of vaccines, including fIPV. These include several needle-free devices  and needle-based devices  .Several of these devices have been tested in clinical studies comparing intradermal fIPV with intramuscular IPV, including the disposable-syringe jet injector Tropis (PharmaJet) in Gambia  and the Other studies have assessed fIPV administration with novel intradermal devices alongside the conventional BCG needle and syringe, allowing a comparison of immunogenicity. In a recent study in Cuba , fIPV adIn the Gambia study , OPV-immAnother clinical study is currently taking place in Gambia to evaluate both the immunogenicity and the programmatic feasibility for use in immunization campaigns of a revised version of the Tropis device in comparison with other intradermal injection methods .Finally, a recent study in Pakistan compared the usability and immune response of fIPV using the intradermal adapter  and a novel intradermal syringe (Star Syringe) with the BCG needle and syringe . It showIntradermal delivery devices have also been positively evaluated in clinical studies of a number of other vaccines. These include influenza vaccination studies using the MicronJet 600 jet injector ; studiesUpon review of available data, the Strategy Committee of the Global Polio Eradication Initiative (GPEI) endorsed in November 2015 the procurement of the intradermal adapter produced by West Pharmaceuticals and the Tropis (PharmaJet) disposable-syringe jet injector for the WHO IPV stockpile for polio outbreak response. These intradermal delivery devices would cost $0.50–$0.85 per injection, which is considerably more than conventional autodisable (AD) syringes ($0.04 per piece). However, a recent analysis conducted by PATH suggested that the dose sparing of fIPV can potentially offset the increased cost of these devices . FurtherCurrently, the use of fIPV administered intradermally is considered an off-label use, and the IPV manufacturers do not intend to license their products for intradermal administration in the foreseeable future. However, it is not uncommon for public health authorities, such as the SAGE or national immunization technical advisory groups, to make recommendations that differ from indications on the vaccine product, especially in emergency situations, such as during yellow fever outbreaks . CountriThe use of intradermal devices requires a separate regulatory clearance  in the United States and CE Mark in the European Union), depending on the country of use. The US Food and Drug Administration has recently added a new requirement that, for vaccines to be relabeled for use with disposable-syringe jet injectors, noninferiority clinical studies of the specific vaccine product using these devices must take place, in addition to the 510(k) clearance for the jet injector itself. The vaccine label can either indicate delivery by jet injection as a class of devices, enabling any 510(k)-cleared jet injector to be used, or specify a particular device . The posUsing multidose vials involves piercing the vial stopper repeatedly with a BCG needle and syringe to draw out each dose. Fractional IPV significantly increases the number of piercings , which could potentially compromise the integrity of the vial stopper. PATH recently conducted two types of tests of vial stoppers used for two WHO-prequalified IPVs following repeated piercing with a 27-gauge needle . Self-seExperiences in several states of India and in Sri Lanka have demonstrated that administering fIPV intradermally in a routine immunization setting can be done safely and effectively, provided there is a proper training program and supervision strategy.The operational feasibility of administering fIPV doses during mass vaccination campaigns in response to outbreaks has also been demonstrated recently in India and Pakistan . An outbreak response campaign conducted in June 2016 in Hyderabad, India  demonstrated the feasibility of successfully planning and implementing a mass vaccination campaign using fIPV in a short period of time . Within To date, the only vaccine wastage data yet available for fIPV vaccination come from the mass vaccination campaigns conducted in India and the study conducted in Karachi, Pakistan, in 2016 . In HydeConsiderable scientific evidence is available to support the use of intradermal fIPV for both routine and supplementary immunization activities. In addition, the development of a range of intradermal devices, such as needle-free jet injectors and needle adapters, show promise for furthering the use of fIPV by improving its usability and the ease of administration in the field. Given the severely limited global supply of IPV, countries are strongly encouraged to consider assessing the programmatic feasibility and trade-offs of introducing IPV using a 2-fractional-dose strategy through their national immunization technical advisory groups or other advisory bodies. This option would not only address the immediate IPV shortage but also serve as an affordable and immunogenic option for routine immunization after global polio eradication has been achieved. One possible strategy is to provide 2 fIPV doses beyond early infancy  because this approach has been shown to result in seroconversion rates among children of >90% .Moving forward, further assessments of the ongoing use of fIPV , as well as more pilot projects, would be helpful to facilitate country discussions and decision making to introduce fIPV. Additional research is also desirable to better understand the role of fIPV in inducing mucosal immunity and long-term immunity to provide further evidence to support the implementation of this strategy.


Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception. Brain function is critically dependent on the correct wiring of neurons during embryonic and early postnatal stages. Extending axons are tipped by a growth cone; a specialized highly motile structure that recognizes secreted and membrane or extracellular matrix bound molecular guidance cues in its local microenvironment. These guidance factors instruct the migration of the growth cone along the correct path to its target region. It is well accepted that the basic cellular and molecular mechanisms underlying the directional pathfinding decisions of the growth cone are attraction and repulsion caused by graded distributions of guidance cues. In a gradient of a guidance cue, a higher concentration might result in a higher receptor occupancy and activation. This in turn will induce a localized intracellular signaling cascade in the growth cone resulting in growth directed towards higher concentrations of the cue or away from it ,2,3,4,5.In this review article, we discuss recent progress on cyclic guanosine monophosphate (cGMP) signaling components implicated in axon branching with a specific focus on sensory axons in the developing spinal cord and hindbrain. In vitro studies on axon branching and growth cone behavior, in which cGMP signaling is modulated, are only briefly mentioned here. For a more detailed description of this field the reader is referred to the reviews listed below. These reviews try to provide an integrated view of how axonal branching might work. However, it is important to keep in mind that the functional in vivo significance of these largely in vitro studies for a mechanistic understanding of axonal branching in vivo is less clear and remains to be established ,10,11,12Xenopus spinal cord neurons and counteracted growth cone collapse [The ubiquitous second messenger cGMP is well known to be involved in a wide range of biological processes ,14. A rocollapse . In cellcollapse ,18,19,20collapse ,22. cGMPcollapse . Therefocollapse .The intracellular signals that are elicited by attractive or repulsive axonal guidance molecules provoke changes of the growth cone morphology which require the action of components of the cytoskeleton and the machinery of vesicle trafficking. These processes might function independently or might affect each other . In vitrDrosophila melanogaster midline by controlling levels of the axonal guidance receptor Robo at the cell surface [It is also conceivable that an increase of local exocytosis might be sufficient to trigger collateral formation of axons, although this has not been studied in detail. Localized exocytosis might regulate cell surface distribution of specific membrane proteins such as receptors for axonal guidance factors. This has been demonstrated for the protein commissureless that regulates axon guidance across the  surface . Related surface . In addition to these studies on growth cone extension, a role for cGMP signaling in the formation of neuronal circuits in vertebrates was also shown by pharmacological manipulations of the soluble guanylyl cyclases that regulate intracellular cGMP levels in neurons ,30,31,32Despite these fascinating and sophisticated in vitro studies on the role of cGMP signaling in growth cones compiled above, our knowledge of cGMP signaling-related axon pathfinding and branching during developmental stages in the nervous system of vertebrates is still fragmentary. However, a number of recent studies that focused on axon branching have shed light on the involvement of cGMP in neuronal circuit building in vivo. The analysis of DRG neuron axon projections into the spinal cord was instrumental in unravelling the function of cGMP in axon bifurcation—a specific form of axon branching. The pattern of sensory axon branching is relatively simple and stereotyped and therefore appeared suitable for a molecular analysis to characterize branching factors . In contSensory axons enter the spinal cord at early embryonic stages at the dorsal root entry zone (DREZ), where they split into ascending and descending arms ,36,37,38It is of importance to note that all sensory axons show more or less the same pattern of branching. This repetitive pattern of arborization is of great advantage for a molecular analysis of branching factors. Moreover, the system is easily accessible to analyze the path of axons by 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI) tracing , by antiThe axonal system described above has enabled the characterization of a cGMP-dependent signaling cascade essential for the bifurcation of sensory axons by applying mouse genetics. This signaling cascade is composed of the ligand C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2  and the cGMP-dependent protein kinase I )—a key effector of cGMP signaling cascades ,46,47,48N,N-dethylamino)-diazenolate-2-oxide dethylammonium salt)/NO (nitic oxide) in real-time imaging experiments with a genetically encoded fluorescent cGMP sensor in cultivated embryonic mouse DRG neurons [Subunits of the nitric oxide-sensitive guanylyl cyclases (NO-GCs)—in some in vitro experiments involving the application of pharmacological reagents shown to be involved in growth cone activities ,47—are n neurons . Taken tInterestingly, the loss of axon bifurcation in DRG neurons in the absence of cGMP signaling does not affect their ability to form normal collaterals in the spinal cord C,D. AlsoA major question is whether the CNP/Npr2/cGKI signaling cascade is also utilized by other projecting axons to bifurcate during development. Generation of reporter mice of CNP and Npr2 and highly specific antibodies to cGKI allowed the identification of additional axon systems that co-express Npr2 and cGKI and the ligand CNP in the region where bifurcation takes place. These investigations showed that cranial sensory ganglia (CSG) neurons  and MTNsNpr2 knockout mice MTN axons do not split in the hindbrain and either grow to the jaw or to the trigeminal motor nucleus  and are, like DRG neurons, pseudo-unipolar. MTN axons initially project from dorsal layers of the mesencephalon ventrally before they pioneer the lateral longitudinal fasciculus to extend further caudally ,59,60,61research ).In summary, it appears that axon splitting regulated by CNP/Npr2 signaling via cGKIα is a unique feature of certain primary sensory neurons in vertebrates.Intracellular cGMP levels are controlled by the rate of its synthesis via guanylyl cyclases and degradation via phosphodiesterases (PDEs) ,72. The Npr3 caused skeletal overgrowth in rodents due to increased levels of CNP in the extracellular space [The mRNA of the scavenger receptor Npr3  is not expressed by DRG neurons but localized in cells—most likely Schwann cells or their precursors—associated with the dorsal roots of the spinal cord ,53. Whilar space ,74, on DNpr2 and cGKI were conditionally inactivated in DRG neurons at early stages using the Cre-driver line Wnt1-Cre. In these mice, sensory axon bifurcation is completely lacking as in global knockouts. Surprisingly, in a number of behavioral tests that examine balance and motor coordination  no deficits were observed indicating that despite the absence of sensory axon bifurcation considerable coordination capabilities are maintained in these mice [The primary sensory representation of the body within the spinal cord is based on the intricate innervation and branching pattern of axons from DRG neurons. This topographic representation of the soma is of fundamental importance for sensory information processing ,76. In tese mice . Npr2 and CNP knockout mice [Npr2 mutant—in which bone growth is reduced—demonstrated deficits in the auditory system [In contrast, loss of axonal bifurcation impairs the rapid response to avoid noxious heat (hot plate test and Hargreaves test), whereas behavioural thresholds and response latencies to cold (acetone-evoked evaporative cooling) or mechanical stimuli (dynamic plantar aesthesiometer) were not affected. Nociception induced by the chemical irritants capsaicin or formalin are impaired by the loss of axonal bifurcation . Consistout mice ,46. In ay system . In summNpr2 mutants. Sensory information from a large number of afferent axons converges in the spinal cord in nociceptive, mechanoreceptive, or proprioceptive fields. Generation of these distinct and overlapping sensory fields in the spinal cord relies on axon collaterals and terminal branches in specific layers. The influence of a loss of bifurcation on the size or shape of termination fields of afferents was recently visualized by transganglionic transport of fluorescently labelled cholera toxin B (CTB). This work showed not only a quantitative reduction of incoming fibers, but also a change in the pattern in the termination fields—most likely caused by an altered terminal branching . For exaThe changes in the termination fields detected by CTB labeling indicate that elaborate compensatory mechanisms are implemented to reorganize neuronal circuits in the absence of bifurcation. The behavioral studies suggest that these compensatory mechanisms might be more active in the proprioceptive than in the nociceptive system.NPR2 gene result in acromesomelic dysplasia type Maroteaux , a skeletal dysplasia with an extremely short and disproportionate stature [Npr2- or CNP-deficient mice show dwarfism [Npr2-deficient mouse mutants might provide a framework for future studies to characterize neurological qualities of human patients with mutations in the Npr2 gene.CNP and Npr2 are also implicated in the process of endochondral ossification which affects long bone growth. Therefore, biallelic loss-of-function mutations including missense, nonsense, frame-shift mutations, insertions and deletions, and splice site mutations in the human  stature ,80,81. M stature ,83. Simidwarfism ,86,87,88