Chemokine-induced leukocyte migration into the vessel wall is an early pathological event in the progression of atherosclerosis, the underlying cause of myocardial infarction. The immune-inflammatory response, mediated by both the innate and adaptive immune cells, is involved in the initiation, recruitment, and resolution phases of cardiovascular disease progression. Activation of leukocytes via inflammatory mediators such as chemokines, cytokines, and adhesion molecules is instrumental in these processes. In this review, we highlight leukocyte activation with the main focus being on the mechanisms of chemokine-mediated recruitment in atherosclerosis and the response postmyocardial infarction with key examples from experimental models of cardiovascular inflammation. Atherosclerosis is a chronic inflammatory condition affecting the medium- and large-sized arteries, characterised by the progressive development of lesions consisting of lipid, fibrosis, and inflammatory cell infiltrate within the tunica media . The proThe established link between hypercholesterolaemia and atherosclerosis led to the characterisation of atherosclerosis as primarily a disorder of lipids, a hypothesis that would dominate thinking for much of the 20th century. However, the last few decades have seen the establishment of inflammation as a key part of atherosclerosis and the important contribution of leukocytes to the initiation and progression of atherosclerotic plaques .This section will focus on the role of leukocytes in atherosclerosis and specifically their migration and trafficking with a focus on the most recent developments in the field.The macrophage is one of the defining cells present in an atherosclerotic lesion in addition to the macrophage-derived foam cell, an enlarged macrophage characterised by accumulation of oxLDL and cholesterol crystals . DerivedMacrophage activation in atherosclerotic lesions is associated with the local release of inflammatory cytokines and reactive oxygen species, both of which contribute to the continued recruitment and activation of leukocytes. Activation is also associated with the release of plaque-destabilizing molecules such as matrix metalloproteinases . Finally−/− (apolipoprotein E knockout) mice leads to significantly marked reductions in lesion formation [−/−ApoE−/− mice [−/− mice with inactivating mutations of M-CSF (macrophage colony stimulating factor) also show marked reductions in both lesion size and macrophage content [Activation of macrophages is dependent upon ligand interactions with pattern recognition receptors . In atheormation , and thi−/− mice , whilst  content .Lymphocytes, particularly T-lymphocytes, are also present in atherosclerotic lesions albeit in smaller numbers than macrophages . HoweverIn turn, T-cell activation plays a role in atherosclerosis with interferon-gamma, the signature cytokine of T helper 1 (TH1) cells, being present in human atherosclerotic lesions and in turn being associated with enhanced cytokine activity, increased macrophage activation, and reduced collagen formation . The rolAtherosclerosis occurs at specific sites in the arterial tree where turbulent flow is present, such as the aortic root and arch, in humans as well as animal models of atherosclerosis with “fatty streak” lesions present from the first decade of life in humans . Whilst Further to this, our understanding of vascular biology has come to demonstrate the important interactions of endothelial cells in trafficking leukocytes to areas of local inflammation and injury. Indeed, the expression of adhesion molecules on their surface allows subsequent interaction between those molecules and leukocyte selectins to enable rolling and extravasation  Figure . As a reen-face immunohistochemical staining in animal studies of atherosclerosis demonstrates the upregulation of VCAM-1 (vascular cell adhesion molecule-1) in turbulent flow sites in hyperlipidaemic animals compared to wild-type animals [−/− mice with hypomorphic variants of VCAM-1 display reduced lesion formation [The expression of several proteins in endothelial cells is linked to the levels of shear stress. Early  animals . In addiormation . Concurrormation . Both VC−/−eNOS−/− mouse which shows increased lesion burden, VCAM-1 expression, and plaque macrophage content [−/− mouse, in conjunction with the overexpression of the rate limiting enzyme (GTPCH1) which is involved in its cofactor synthesis, to prevent eNOS uncoupling, shows markedly reduced lesion burden [Conversely, in sites of laminar flow, there is upregulation of several “athero-protective” genes such as eNOS , with the subsequent nitric oxide release serving to combat oxidative stress as well as to directly limit VCAM-1 expression through the inhibition of NF-κB . Indeed, content . Conversn burden .Thus, it appears that in endothelial sites of turbulent flow, there is upregulation of adhesion molecules with enhanced upregulation in the presence of hyperlipidaemia and relative downregulation of endothelial genes important in preventing atherogenesis. This initial process and the subsequent recruitment of leukocytes as well as lipid into the subendothelial spaces of turbulent flow sites lead to the creation of a local inflammatory environment, where lipid is oxidised and leukocytes are activated, which is critical in the development of lesions .At the sites of inflammation, leukocyte attraction and retention not only requires the local expression of endothelial adhesion molecules but also the activation of leukocytes and subsequent conversion into a high affinity state. This is typically achieved through the local release of chemical mediators, principally the chemokines (chemotactic cytokines), from local tissue-resident cells .In the initial lesion, the entrapment of LDL (low density lipoprotein) and its subsequent oxidation leads to localised inflammation and the release of chemokines with other inflammatory mediators; this synergises with shear stress-induced expression of adhesion molecules to recruit leukocytes which become activated by and then contribute to the inflammatory environment .α. Upon release, chemokines bind to glycosaminoglycans on cell surfaces to establish local gradients that are able to activate and then direct leukocytes to specific sites [Chemokines are 8–12 kDa proteins that signal through their respective G-protein-coupled receptors (GPCRs). They are released from endothelial cells, platelets, macrophages, and lymphocytes following activation by inflammation and cardinal inflammatory mediators, such as TNF-ic sites .Chemokines and chemokine receptors are significantly implicated in atherosclerosis with chemokine receptors known to be expressed within animal and human plaques and their expression being correlated to progression .−/−CCR2−/− murine model which shows significant reductions in macrophage content and plaque content compared to ApoE−/− mice [−/− displays striking reductions of at least 50% of lesion area as well as greater reductions in macrophage content and an increase in smooth muscle content [Although the chemokine system displays a great deal of redundancy, the development of chemokine receptor knockouts in atherosclerosis murine models has given insight into their role. This was first demonstrated in the ApoE−/− mice . Additio content .−/− (LDL receptor) mice for 14 weeks resulted in significant lesion area reduction as well as a reduction in CCR2 expression suggesting an anti-inflammatory and antiatherogenic effect [−/− mice [In vivo antichemokine strategies have also been successful in reducing plaque burden. The antagonism of RANTES  with met-RANTES in LDLRc effect . The use−/− mice .−/−CCR1−/− murine model demonstrating an increase in lesion area with increased levels of T-cell infiltration, providing further evidence of the contribution of T-lymphocytes [However, chemokines do not appear to be universally proatherogenic, with the ApoEphocytes .Chemokines are also known to play an apparent role in the retention of leukocytes within the plaque, although this has not been as comprehensively studied as the process of leukocyte recruitment in atherosclerosis. For example, the chemokine CX3CL1 promotes macrophage adhesion to smooth muscle cells (SMCs) within coronary artery plaques and its expression is elevated in hyperlipidaemia. Conversely, a reduction in CX3CL1 expression reduces macrophage adherence to SMCs .Perhaps most striking is the apparent role of chemokines in leukocyte migration in regression models of atherosclerosis. In the surgical model, whereby regression in an affected artery is induced by transfer to a normo-lipidaemic animal, there is a reduction in VCAM-1 levels in the atherosclerotic artery but, conversely, an increase in CCR7 levels. Further to this, when recipient animals were treated with antibodies against the CCR7 ligands, the regressing lesions were markedly impaired, with retention of leukocyte content seen as well, suggesting a failure of migration .Finally, in addition to the principal role of chemokines in leukocyte migration in atherosclerosis, other molecules such as chemotaxins may also play a role. Leukocyte cell-derived chemotaxin 2 has recently been shown to correlate with atherosclerotic lesion burden and macrophage content in human tissue samples , and furOne of the more recent developments in atherosclerosis research has been the production of murine models that facilitate tracking of myeloid cells thus providing new insights into myeloid cell biology beyond the work previously discussed regarding the role of chemokines and shear stress-linked regulation of genes.−/− animals which demonstrates a dominance of donor myeloid cells in the plaque [It has been established, for instance, that the macrophages and foam cells in murine experimental plaques are mainly recruited from circulating monocytes as opposed to local resident cells . An exame plaque .lo/Ly6Chi monocytes preferentially migrate to the sites of acute inflammation whereas the CX3CR1hi/Ly6Clo population does not [It has also been possible to elucidate the role of different macrophage subtypes. The creation of a murine model expressing GFP under the CX3CR1 promoter revealed the existence of low-expression and high-expression populations with expression of CX3CR1 being negatively correlated to the expression of Ly6C and Gr-1. It was subsequently found that CX3CR1does not .−/− mice, when induced through diet to develop hypercholesterolaemia, is dominated by the CX3CR1lo/Ly6Chi population. Indeed, in this model, the CX3CR1lo/Ly6Chi population doubled every month on a high-fat diet and is correlated with lipid levels; conversely, the monocytosis of this population is attenuated by statin treatment [lo/Ly6Chi monocytes proving not only to be a reservoir but also an active source of monocyte generation [Further to this, it is now understood that the peripheral blood monocytosis that develops in ApoEreatment . Additioneration .lo/Ly6Chi population is not seen in animals on high-fat diet when CCR2 is knocked out for instance [lo/Ly6Chi monocytes on CCR2 for migration [This work has also been applied to further demonstrate the role of chemokines in leukocyte migration in atherosclerosis. The monocytosis of the CX3CR1instance . In addiigration .lo/Ly6Chi monocyte population is now known to express higher levels of PSGL-1 (P-selectin glycoprotein ligand-1), an adhesion molecule, compared to CX3CR1hi/Ly6Clo monocytes. In turn, the PSGL-1 null mouse displays a reduced migration of CX3CR1lo/Ly6Chi monocytes to atherosclerotic plaques with a concordant reduction in lesion area [It has also offered further insights into the role of adhesion molecules. For example, the CX3CR1ion area .GFP model previously discussed, enables the study of monocyte fate as well as initial trafficking and recruitment [Finally, a murine model expressing GFP under the control of the human CD68 promoter has recently been created which drives GFP expression in all monocytes of the blood, spleen, and bone marrow. Most promisingly, these monocytes retain high levels of GFP expression for at least 72 hours after differentiation into macrophages and so, unlike the CX3CR1ruitment .GFP ApoE−/− mice, where the expression of GFP had no significant effect on the atherosclerosis process and allowed clear visualisation of plaque macrophages, with quantification by GFP correlating with traditional macrophage quantification methods [−/− with established plaques demonstrated clear visualisation of GFP+ cells 72 hours posttransfer further confirming the ongoing recruitment of peripheral monocytes as the source of the plaque macrophage population [Indeed, this model has already been utilised in atherosclerosis research with the generation of CD68 methods . Adoptivpulation . In contAccording to the American Heart Association, myocardial infarction (MI) and congestive heart failure (CHF) mortalities are the major contributors of the cardiovascular diseases that are the leading causes of death in the Western world . MI descFactors that increase the lifetime risk of an acute  or chronic (CHF) coronary syndrome, which manifest over decades, are a high-lipid diet (atherosclerosis), low levels of exercise, chronic diseases (diabetes), smoking, and genetic predisposition  . The aveCardiovascular disease was first described in 1772 by Herberden ; symptomα , and IL-1β/IL-18 stimulates neutrophil recruitment within 12–24 hours post-MI. The positive self-feedback loop of CCL8 secretion recruits further neutrophils to the infarct for the phagocytosis of necrotic, autophagic, or apoptosed cardiac tissue [The most frequent coronary vessel occluded in MI is the left anterior descending artery (LAD) which nourishes the left ventricle (LV), and the ischaemia that follows impairs the endothelial vascular integrity leading to leukocyte infiltration . The carc tissue . Neutropc tissue . Neutropc tissue .hi monocytes (recruited via CCR2) and the prohealing Ly6Clo monocytes (recruited via CX3CR1), differentiating to various subsets of macrophages within the classically activated M1 and the alternatively activated M2 phenotypes [The immune response to acute MI can be divided between the inflammatory phase at days 1–4 and the healing phase from days 5–14 from which point the scar matures  Figure . Certainenotypes . In humaenotypes .hi monocytes from the blood pool via their receptor CCR2 and initiates HSC (haematopoietic stem cell) progenitor proliferation in the bone marrow [With the innate immune response underway, tissue-resident macrophages are proliferating under the control of M-CSF, from activated endothelial cells, fibroblasts, and macrophages, and secrete increasing levels of CCL2. CCL2 stimulates the recruitment of the Ly6Ce marrow .β3-adrenergic receptors in the bone marrow, reducing levels of CXCL12 (the “monocyte progenitor egress” inhibitory cytokine) and allowing HSC relocation to the spleen for proliferation and maturation [Furthermore, MI activates the neuro-humoral axis via sympathetic innervations of adrenaline secretion for turation . Limitinturation . Splenicturation .hi monocytes to the infarct continues the acute phase response and the efferocytosis of dead cardiac tissue including the apoptotic neutrophils, preparing the infarct site for new ECM and collagen deposition by fibroblasts and Ly6Clo monocytes. Prolonged inflammation via the Ly6Chi monocytes is associated with dilative remodelling (LV thinning) and systolic dysfunction, ultimately leading to heart failure when the inflammatory phase is not resolved and Ly6Chi cells repopulate the LV weeks to months postinfarct healing [Extravasation of Ly6C healing .−/− mice [−/− could be due to CCR1 and CCR5 low affinity for CCL2 released by the infarct.The mechanisms of monocyte recruitment in MI were first challenged by Kaikita et al. using CCR2−/− mice . The aut−/− animals have impaired necrotic cell clearance, collagen deposition, and angiogenesis with the predisposition for cardiac rupture and immediate death [Further studies have shown that a balance between proinflammation versus healing is key for cardiac remodelling as CCR2te death . Overactte death .lo monocyte populations, with decreased peripheral levels compared to the Ly6Chi populations (in Nr4a1−/−), Hilgendorf et al. [lo monocytes is amplified by a Ly6Chi monocyte to Ly6Clo macrophage conversion, with Nr4a1 levels fluctuating between the two cell phenotypes with high levels dampening the proinflammatory macrophage secretions.Building on the work of Hanna et al. , which df et al.  identifihi monocyte recruitment but its subsequent differentiation to the infarct macrophage populations. Additionally, Ly6Clo monocytes are also recruited to the infarct from the blood via CX3CL1 release through endothelial cells [lo cells as well as TGF-β, a stimulator for ECM gene expression by cardiac myofibroblasts. Angiogenic factors are also secreted by the Ly6Clo cells such as VEGF  to restore oxygen supply by neovascularisation and to establish the granulation tissue for scar formation/maturation [β (transforming growth factor beta) signalling and TCRP6  expression to switch from cardiac fibroblasts [The previous finding that ACE inhibitors reduced the leukocyte populations can now be explained by not just inhibiting Ly6Cal cells . With thal cells . Phagocyturation . Noncontroblasts .β) [From a clinical perspective of leukocyte migration in atherosclerosis, the chemokine system represents a particularly excellent candidate for exploring therapeutic options. Whilst the chemokine system displays a great deal of redundancy, there are clear and powerful effects seen in specific murine knockouts of key chemokine receptors which might be replicated. Indeed, the chemokine system could be targeted through several different avenues, such as classical GPCR antagonists, for example. Due to the important role of chemokines in wound healing and infection control, long-term therapy would likely be associated with adverse effects. However, utilising chemokine inhibition to stabilise atherosclerotic plaques in particular circumstances could well provide clinical benefit. Such examples might include delivery of therapy to stabilise plaques following myocardial infarction to prevent further rupture or vessel stenosis or delivery of therapeutics in transient ischaemic attacks to reduce stroke risk due to carotid artery atherosclerosis. With several drug targets against chemokines already identified in drug discovery programmes, this field bears considerable future potential . In addiβ) , the mecThe authors declare that they have no conflicts of interest.


Expressed human milk (EHM) feed preparation areas represent a potential source of unintentional nosocomial infection. Daily disinfection of environmental surfaces remains an essential intervention to mitigate nosocomial infections. The inefficiency of conventional cleaning and disinfection contributes to an increased risk for the acquisition of multi-drug resistant pathogens. “Non touch” technologies such as the pulsed xenon ultraviolet (PX-UVD) light device have documented sustained reduction in surface bacterial colonization and reduced cross contamination.2) in feed preparation areas was evaluated following its implementation as standard care. A quasi-experimental study was performed documenting bacterial colonization from 6 high risk feed preparation areas in a community care hospital in South Africa. Pre and post conventional cleaning neutralizing rinse swabs were collected fortnightly over a 16 week control period prior to the introduction of the PX-UVD and compared to a matching set of samples for the PX-UVD period.The impact of a PX-UVD on surface colony forming units per square centimeter (cfu/cm2) compared to the corresponding PX-UVD period (50 cfu/cm2). Sub -analysis of both the Pre-clean Control: Pre-clean PX-UVD counts as well as the Post-clean Control: Post-clean PX-UVD counts noted significant improvements (p < 0.001). A statistically significant improvement was noted between pre-and post-cleaning total surface bioburden following exposure to the PX-UVD (p = 0.0004). The introduction of the PX-UVD was associated with a sustained reduction in the pre clean bioburden counts with a risk trend (per week) 0.19, .A 90% reduction in total surface bioburden was noted from the control period (544 cfu/cmThe use of a PX-UVD as adjunct to standard cleaning protocols was associated with a significant decrease in surface bioburden. The study demonstrated the inefficiency of conventional cleaning. Persistence of potentially pathological species in both periods highlights current health sector challenges. Expressed human milk (EHM) feed preparation areas remain an integral aspect of neonatal intensive care as well as pediatric critical care units, and are common place in any hospital setting. These areas also represent a key source of infection and contamination resulting in unintentional nosocomial infections , 2.The South African National Department of Health (DOH) has strict procedural requirements for any designated feed preparation area including the use of sterile gowns and gloves during the preparation of feeds by trained individuals in designated well marked areas. The Netcare private hospital group has additional standard operational procedures (SOP) for the daily disinfection of feed preparation areas as well as the safe preparation, storage and handling of expressed human milk. Although daily disinfection of environmental surfaces remains an essential intervention to mitigate nosocomial infections , the ineThe pulsed xenon ultraviolet (PX-UVD) light device is a “non touch” ultraviolet C (UV-C) emitting technology designed for the hospital setting. Each pulse from the non-mercury Xenon flash lamp releases approximately 505 J of energy into high intensity broad-spectrum UV light, with a narrow band concentration within the UV-C spectrum . The ger2) within neonatal and pediatric EHM feed preparation areas at Netcare Blaauwberg hospital.The aim of this study was to evaluate the effect of a pulsed-xenon ultraviolet portable device (PX-UVD) as compared to standard care on surface colony forming units per square centimeter , a 16 bed pediatric ward and a 16 bed maternity ward.The NICU, maternity and pediatric wards actively participate in the baby friendly initiative promoting human milk exclusivity. The NICU utilizes a multi counter dedicated expressed human milk (EHM) feed preparation area for the processing of stored fresh and frozen EHM. The maternity and pediatric wards have a dedicated single counter feed preparation area. Reconstitution of dry milk formulae only occurs within the pediatric and maternity wards on strict prescription of the attending pediatricians.A quasi-experimental study was conducted from June 2015 until February 2016. The study was approved as a nonhuman-subject, quality-improvement study by the Netcare research operations committee and the University of Stellenbosch ethics committee.™ Transwab ®). The six high risk areas identified included:, the NICU prewash EHM bottle area, the NICU post-wash EHM bottle area, the NICU EHM preparation area, the NICU fridge door handle and the single counter surface within the feed preparation areas of both the pediatric and maternity wards.Environmental surface bioburden was evaluated by collecting pre – and post cleaning surveillance swabs from 6 surfaces in 3 feed preparation areas using pre-immersed neutralizing rinse swabs  × 10 cm area for each surface as per the recommendation of the resident clinical microbiologist.Following pre clean sampling, the area was cleaned as per the facility’s SOP. The facility’s SOP for daily terminal cleaning of working surfaces in the feed preparation areas involves initial cleaning with soap and water using commercially available disposable cloths, followed by disinfection with a suspension of Troclosene Sodium (NaDCC) at 500 ppm (ppm). Cleaning of the fridge door and handle is a specifically allocated area and includes the aforementioned protocol in addition to weekly cleaning of the inside of the fridge and monthly defrosting. One designated trained multi-shift cleaning team is allocated to this duty on a continuous basis. The area is then allowed to air dry for 1 h after which post cleaning swabs were taken from the same allocated areas.Cleaning staff and nursing staff were blinded to the details of the study as well as to the timing of the swabs, allocated areas and frequency of sampling. The facility’s head of infection control and resident microbiologist remained blinded to the sample results for the duration of the study.A total of 108 CONTROL samples were collected fortnightly over a 16 week period prior to the implementation of the PX-UVD on week 17 of the study. The introduction of the PX-UVD to the standard cleaning protocol involved the daily cleaning of the allocated feed preparation areas as per facility’s SOP including an air dry period for 1 h. Thereafter the PX-UVD was placed on either side of each of the 3 feed preparation areas for a 5-min treatment cycle, as per manufacturers recommendations. Post cleaning swabs were taken immediately after exposure to the PX-UVD. A matching 108 PX-UVD samples was collected over the ensuing 16 weeks.2). The colonies cultured, included both natural environmental contaminant species as well as potentially pathogenic species, were then transferred to agar plates for further organism identification.The pre-immersed neutralizing rinse swabs (NRSII™ Transwab ®) were immediately collected and transported by Pathcare laboratory services in a temperature regulated environment for processing at their off-site facility. Each swab container underwent mixing by vortexing 1 ml of neutralizing rinse solution which was then placed on a total viable count (TVC) Petrifilm (3 M Rehydratable film method) agar. Petrifilm agars were then incubated at 35° ± 2 °C for 48 ± 3 h. The total viable count was then quantified into number of colony forming units per square centimeter (cfu/cm) was received on loan from Kiara Healthcare for the duration of the 4-month study period. Floor plans, counter heights, and room dimensions were relayed to the manufacturer. The optimal efficacy for the device was mathematically modelled based on spectrometer data and the location and size of the target areas. The resulting recommendation of two treatment cycles of 5-min per side of each allocated feed preparation area was determined to ensure maximum counter exposure with no shadow areas.A single PX-UVD  of the 6 counter surfaces in the pre and post cleaning phases. Statistical analyses was performed using the NCSS statistical analysis package . NCSS, LLC. Kaysville, Utah, USA.)Total surface bioburden was calculated as the sum of the viable colony count  compared to the corresponding PX-UVD period (50 cfu/cm2). Pre cleaning surface bioburden significantly improved from 244 cfu/cm2 in the CONTROL period to 44 cfu/cm2 in the PX-UVD period with a geometric mean risk ratio 0.11, . Similarly, the post cleaning surface bioburden significantly improved from 300 cfu/cm2 in the CONTROL period to 6 cfu/cm2 in the PX-UVD period with a geometric mean risk ratio 0.04, . Individual counter surface data during the CONTROL period noted higher average surface bioburden within areas of the NICU, most noteworthy the post-wash EHM bottle area and the EHM preparation area recorded higher surface bioburden counts post conventional cleaning. . ) a statistically significant improvement was demonstrated between the pre cleaning total surface bioburden and the post cleaning total surface bioburden following exposure to the PX-UVD ), including complete eradication of detectable bacteria in weeks 18 and 28.The graphical representation of the CONTROL period Fig.  demonstrTwenty three pathological organisms were identified during the control period in comparison to the 5 identified during the PX-UVD period Table .Table 2OExpressed human milk, particularly within the confines of the high-risk environment of the neonatal ICU, represents a critical irreplaceable aspect of the care for these highly vulnerable and immunocompromised infants. Ensuring a sterile dedicated environment for the processing and handling of EHM cannot be overemphasize. Despite our compliance with the South African Department of Health’s and facility’s recommendations for surface disinfection, this study highlighted the inefficiency of conventional cleaning on both natural environmental contaminants and potentially pathogenic species. The significantly higher total surface bioburden counts and increased post clean total surface bioburden counts during the control period invariably contributed to the diversity of potentially pathogenic isolates identified during this period.The introduction of a “no-touch” PX-UVD as an adjunct to the facility’s conventional cleaning SOP was associated initially with a dramatic reduction in both the pre and post clean total surface bioburden. Subsequently, a sustained reduction in the pre clean surface bioburden counts together with a stabilization and consistent improvement between the pre- and post cleaning surface bioburden, culminated in a statistically significant reduction in pre and post cleaning total surface bioburden for the PX-UVD period. The susceptibility of both environmental contaminants and potentially pathogenic organisms to the germicidal effects of UV-C exposure remains cautiously reassuring of the potential long term sustained effects of PX-UVD.Staphylococcus aureus with documented sensitivity to UV-C [The relative dominance of potentially pathogenic gram-negative isolates, as opposed to gram-positive organisms such as Clostridia difficile and Methicillin-resistant  to UV-C –7, was p to UV-C  and docu to UV-C .The limitations of this study include the relatively small study numbers, limited study duration and the lack of variability of performing a single institution study. We did not evaluate the potential long term cumulative suppressive effects following the introduction of the PX-UVD as well as its impact on both environmental and potentially pathogenic organisms; nor the potential impact of a lower surface bioburden and its effect on nosocomial infection rates.Despite these limitations, as a quality improvement study, several strategies have been strongly recommended and subsequently implemented. Expressed human milk feed preparation areas have been deemed high priority areas. The facility’s SOP has been amended to include the conversion to a commercially available quaternary ammonium disinfection solution to negate the potential risk of over-dilution of NaDCC, nonwoven microfiber spunlace cloths have replaced the commercially available disposable cloths for disinfection and the specialized cleaning teams have been re-educated emphasizing on key impact measures such as disinfectant contact time. In addition, a quality assurance monitoring system using adenosine triphosphate (ATP) bioluminescence was introduced to evaluate cleaning practices within the EHM feed preparation area, providing feedback to the specialized cleaning teams. The acquisition and permeant inclusion of a PX-UVD as standard care has been strongly recommended.The use of a PX-UVD as an adjunct to the facility’s standard cleaning protocols within the EHM feed preparation areas was associated with a significant decrease in surface bioburden. Future long term studies are envisioned to evaluated the relationship of a reduced surface bioburden and its impact on nosocomial infection, particularly within neonatal ICU.


Here, we applied whole exome sequencing (WES) on 19 trios from singleton Saudi families with ASD. We developed an analysis pipeline that allows capturing both de novo and inherited rare variants predicted to be deleterious. A total of 47 unique rare variants were detected in 17 trios including 38 which are newly discovered. The majority were either autosomal recessive or X-linked. Our pipeline uncovered variants in 15 ASD-candidate genes, including 5  that have not been reported in any human condition. The remaining variants occurred in genes formerly associated with ASD or other neurological disorders. Examples include SUMF1, KDM5B and MXRA5 (Known-ASD genes), PRODH2 and KCTD21 (implicated in schizophrenia), as well as USP9X and SMS . Consistent with expectation and previous studies, most of the genes implicated herein are enriched for biological processes pertaining to neuronal function. Our findings underscore the private and heterogeneous nature of the genetic architecture of ASD even in a population with high consanguinity rates.Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with genetic and clinical heterogeneity. The interplay of  This is best exemplified by ASD, for which delivering a near complete genetic picture is hindered by many factors. Examples of such factors include: (i) extensive clinical and genetic heterogeneity 2; (ii) the absence of generalizable genetic risk factors as most of the mutations are extremely rare or private in nature; (iii) the variability in diagnosis and data analysis methodology; and finally (iv) the precise function of many of ASD-candidate genes and therefore their possible impact on the central nervous system remains largely undetermined4. All these factors present a substantial challenge, yet incremental progress has and continues to be made towards identifying ASD-risk loci.Delineating the genetic architecture of Autism Spectrum Disorder (ASD) is like finding the way in a labyrinth. Despite the rapid advancements in genetic methods, our ability to identify a common pathway for neuropsychiatric disorders with a therapeutic or diagnostic potential is lagging behind5, as well as recurrent CNVs involving genes like, NRXN1, SHANK3 and PARK26. However, a causal relationship is often hard to prove for a number of reasons: (i) such events normally span large regions making it difficult to discern the role of single genes in the disorder; (ii) apparently identical alterations have variable phenotypic outcomes or expressivity; (iii) some of these changes are observed in typically developing individuals; (iv) very low rate of replication, as recurrent individual CNVs were observed only in less than 1% of the cases7.Over the past 15 years, efforts were centered on interrogating the role of structural alterations through the study of chromosomal abnormalities and copy number variations (CNVs) in ASD individuals. Key advances have emerged from such studies, for instance, the identification of cytogenetic abnormalities at multiple loci with chromosomes 15q11–13 and 16p11.2 being the most frequentde novo point mutations. This move was triggered by the observation that potentially risk-conferring structural alterations occurred only in (1–20%) of the cases7 and that they probably require other genetic or non-genetics factors for the development of the disease. The increasing availability of whole-genome and –exome sequencing platforms to researchers have accelerated the identification of additional risk factors in previously recognized as well as unrecognized ASD-associated genes18. However, most of these studies have focused on the role of either de novo or inherited variants20 and much fewer were designed to interrogate both types22. As there is a growing appreciation for the important part transmitted and de novo mutations play in ASD, more studies assessing both types of events within a given cohort are needed to understand their precise role.In the more recent years and with the advent of next-generation sequencing (NGS) technologies, the focus shifted towards investigating the role of inherited and 17 as are sporadic (de novo) mutations in families with apparently “unaffected” parents24. We therefore, devised an analysis pipeline that enabled us to detect de novo as well as inherited variants in autosomal or X-linked genes , USP9X and SMS , TRIM9 and KDM5B (known-ASD genes), and others such as AGL and MOGS .In this study, we implemented whole exome sequencing (WES) in an attempt to identify risk genes/rare variants in a cohort of 19 case-parent trios from singleton families with ASD from Saudi Arabia. Enrichment for inherited causes are predicted in highly inbred populationsSupplementary Table Ajk statistics and shared homozygosity were more confirmative than the ones generated by KING program.Reported familial relations history was assessed with three different relatedness tests (described in the methods). The results confirm correct parenthood for all our trios  developed in this study allows for comprehensive investigation of the genetic architecture of ASD Fig. . It is dde novo, 4 were autosomal dominant, 21 were found in X-linked genes, and the remaining 19 were inherited in an autosomal recessive manner , while the remaining 38 variants were novel. Altogether, we have identified 47 rare variants in 47 different genes including 15 ASD-candidate genes (Tables Using the 4 main inheritance models (refer to methods) a number of variants was detected in each sample except two (ASD-52 and ASD-55). From the variants that have passed all filtering steps, 44 were considered to be deleterious by at least one prediction tool and were confirmed as being true positives for presence of the variant by Sanger sequencing. The majority of the validated variants were missense, while the remaining were either frameshifts, splice-site changes or nonsense and only one small deletion was detected Fig. . As for ner Fig. . Only 9 De novo germline mutations arising spontaneously during meiosis have long been known to confer risk of ASD. This knowledge was inferred largely from studies assessing de novo CNVs in ASD cases from simplex or multiplex families31, however, structural variants seem to account for a small fraction of the cases34, shifting the focus to de novo point mutations and their contribution to risk, which is a line of enquiry we and others have perused24.de novo SNVs. These confirmed de novo events were identified in different genes including one with previous association with ASD: two of these events were predicted to result in a premature truncation, and the remaining was a missense rare variant (p.G209R) affecting PRODH2, found in proband ASD-58. The two nonsense variants were: (i) (p.Y755X) affecting KDM5B identified in ASD-19 and (ii) a frameshift insertion of two adenine bases creating a premature stop codon (p.K278Efs*5) in MOGS that was detected in proband ASD-24  were found to carry rare riant p.G09R affecde novo mutations, only a fraction of ASD cases could be accounted for by these mutational events17, while cases bearing inherited events couldn’t be fully explained by this class of genetic alteration. The high heritability of ASD15 underscores the need to explore transmitted variants that are often overlooked in many of the existing exome studies, which are mainly focused on de novo variants and not designed to capture inherited variants24. Investigating both de novo and inherited variants has become a favored approach in recent next-generation sequencing based ASD studies18.Despite the excess and well-recognized importance of Considering the high rate of consanguinity in the Saudi population, a recessive model was implemented under which a total of 19 rare variants were identified in different genes Table .APC2, AGL, NEB and SEMG2). Two of the variants were observed in ASD-9 occurring within a splice site region of APC2 and AGL. One missense variant affecting NEB was found in ASD-39 and a nonsense variant within SEMG2 was found in ASD-66 . By only considering rare variants that were transmitted to the proband from one heterozygous parent, 4 rare variants were confirmed in 4 different genes . The remaining variants existed in genes already reported in ASD or other neurological disorders.By setting our analysis approach to identify inherited variants in X-chromosome genes, we detected and confirmed a total of 21 rare variants  and each was found in a different gene . We could not comment on whether our detection yield is comparable with other studies or not, due to the lack of published ASD trio exome studies of similar design using the same platform. However, Jiang and colleagues conducted a relatively similar study (utilizing an Illumina platform) in which they have reported successfully detecting medically relevant variants in 50% of their ASD trio families using whole-genome sequencing (WGS)10.Dissecting the genetic architecture of an otherwise highly complex disorder such as ASD, comprising diverse forms of genetic alterations , requires employing different approaches. Ideal approaches would be those permitting genetic investigation of ASD cases, in which a polygenic model is assumed (or strongly suspected)de novo events, whereas rare variants in autosomal or X-linked genes were detected in equal number of probands. While most of the previous studies concerned with sporadic autism have focused on assessing the role of de novo variants under the hypothesis that such lesions are more likely to confer risk to ASD than inherited events38, we, on the contrary, observe enrichment for inherited variants. This is unsurprising given the highly inbred nature of the Saudi population in which common ancestry is suspected even in apparently non-consanguineous unions as parents are often unaware of consanguinity in previous generations. Such observation is common in Middle Eastern and North African populations39.Missense variants constitute the most frequently encountered type of variation (77%) in this study followed by loss of function variants  Fig. . Of the SUMF140, HSPBP141, TRIM942, DUSP324 and BOC43. WES detected de novo variants in three genes (Table PRODH2)45 and the other two were protein-truncating variants. One was identified in KDM5B. This gene was previously associated with ASD9 and non-syndromic intellectual disability (ID)46 whose function is related to cell cycle control and neural cells differentiation47. The other was a frameshift insertion of two adenine bases creating a premature stop codon in MOGS. This gene encodes an endoplasmic reticulum glucosidase involved in normal immune function48.In our study, 32 of the detected variants were observed in genes previously reported in ASD (either in Autism databases or in literature) Table . For insMAN1B1, a gene causally linked to non-syndromic intellectual disability50, and another was found in DNAJC13 previously reported to be mutated in patients with Parkinson’s disease52. Two variants were identified in genes with possible, but not confirmed association with mood disorders: CRY155 and NT5DC157. Interestingly, patient (ASD-21) harboring NT5DC1 missense variant displayed symptoms of depression and poor appetite around the age of 5 years. Amongst the autosomal transmitted variants, six were predicted to disrupt a group of genes involved in brain development and/or function namely TRIM9, BOC, SSTR3, NGF, FGF5 and CELSR265, two were found in genes related to protein homeostasis (KCTD2166 and HSPBP167) and the remaining variants were identified in genes involved in diverse biological process including cell signaling (DUSP3)68, inflammatory responses (NLRP2)69, olfaction (OR6C65)70, maintenance of DNA integrity (CEP152)71, and stabilization of the extracellular matrix (ITIH2)72.By applying the autosomal recessive model, we validated variants in 19 genes Table . One misMXRA5, a gene that was reported to be mutated in three multiplex ASD families19. A deletion of 12 nucleotides was detected within exon 2 of AVPR2, defects in this gene cause X-linked congenital nephrogenic diabetes insipidus (NDI) and where detected in dizygotic twins with NDI and ID73. One putative splice site variant was found in PDK3, a gene that encodes an enzyme involved in the shift of energy production site from the mitochondria (oxidative phosphorylation) to the cytoplasm (glycolysis) reported in cancer cells74. The same gene was recently reported to be mutated in a single family with Charcot–Marie–Tooth disease75. However, the patient (ASD-38) reported here to carry PDK3 splice site variant is not affected with Charcot-Marie-Tooth disease, nor does he suffer from any type of neuropathy. Moreover, of the variants detected on the X-chromosome, four were located in genes involved in brain function/development  and two were present in genes regulating splicing and/or transcription (HTATSF181 and GPKOW83). Additional rare variants were identified in genes involved in: (i) the hydrolysis of angiotensin II (ACE2)84 or sulfate esters (IDS)85 and (ARSH)86; (ii) or in the ubiquitination and proteasomal degradation of creatine kinase-B (ASB-9)87. Furthermore, two missense variants were detected: one in ATP2B3 and another in RPS6KA6. Both genes participate in signal transduction pathways, however only ATP2B3 was linked to a human disease89. The remaining variants were located in genes of largely unknown function such as  or in cancer-associated genes namely; INTS6L91 and SSX392.The X-linked model revealed 21 different variants . Aside from the disruption of neuron-specific functions, defects involving basic cellular processes (presented here and elsewhere) represent another commonly encountered theme in ASD, reflecting the clinical and genetic complexity and variability of this conditionNLRP2 and MOGS) with established roles in the immune system. The former encodes for NLRP2 protein that plays a vital role in astrocytes innate immunity69 and was found to be mutated in a rare type of imprinting disorder known as Beckwith-Wiedemann syndrome93. The latter gene (MOGS), on the other hand, encodes an endoplasmic reticulum glucosidase involved in normal immune function and is causally linked to congenital disorders of glycosylation type II B clinically characterized by developmental and neurological defects48. It is worth mentioning that immune dysfunction has been documented in ASD and emerging studies propose a role for the immune system in the pathophysiology of autism. In support of this notion, typical behavior and neuropathological symptoms of human ASD were successfully recapitulated in mice born to immune-activated (infected) mothers94.One of the interesting findings emerging from this study is the identification of two genes (de novo variants (multiple hit) in different genes (genetic heterogeneity), including genes associated with other neuropsychiatric disorders (pleiotropy) or involved in diverse biological processes beyond synaptic function (molecular diversity), shape the risk to ASD95Another key finding demonstrated here, is the genetic variability not only across trios but within each proband. Defects in genes involved in diverse biological processes were observed in most probands and only few  that were enriched for variants affecting neuronal function/development. Our findings are in keeping with a growing number of reports suggesting a genetic model of autism whereby cumulative contribution of multiple inherited and et al. exome sequencing study, aimed at identifying inherited SNVs in a cohort of consanguineous/multiplex families with ASD including two Middle Eastern multiplex families (one from Saudi Arabia and one from Kuwait)17, the genetic characteristics of ASD in Arab populations remain largely unexplored. This work represents, to the best of our knowledge, a comprehensive exome analysis of trios with ASD from an Arab population. Our primary goal was to identify de novo or rare inherited coding variants of potential clinical relevance through applying WES on case-parent trios from singleton families. Among the advantages of implementing this approach over WGS is that it generates fewer results and therefore is relatively less analytically challenging. Although this approach enabled us to generate a prioritized shortlist of potential deleterious variants, association and extensive functional studies are necessary to identify the disease causing ones with more certainty. Also it is important to consider the possibility of missing candidate variants within coding regions and the fact that neither common variants nor non-coding regions were considered here. In spite of the small number of cases investigated here; our current study is useful in terms of contributing a small, albeit rich data set, revealing new ASD candidate genes that may shed light on potential diagnostic and therapeutic targets.Apart from Yu Patient recruitment and all experimental protocols used in this study were in compliance with the Declaration of Helsinki and were approved by the institution’s relevant committees; Internal Review Board (IRB), Research Ethics committee and Basic Research Committee at King Faisal Specialist Hospital and Research Center. Written informed consent was obtained from subjects before enrollment in an IRB-approved protocol  RAC#2080001).96, a common lineage is suspected even when couples regard themselves as unrelated as they are often unaware of consanguinity in distant generations97. Diagnosis of ASD was based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) criteria  and ADI-R, ADOS . Both parents were present for the interviews and assessment. All selected cases did not have symptomatic ASD secondary to known genetic or metabolic disorders . Available clinical and demographic information is summarized in Supplementary Table A total of 19 simplex (singleton) families were selected for the study . Venous blood samples from parents and affected child were obtained for both DNA and RNA extraction. Of the recruited families, 9 were from consanguineous marriages and 10 were not. Non-consanguineous families were included based on the rationale that in populations with high levels of consanguinity/endogamy All probands were negative for any copy number variants (CNV) in previously reported ASD-associated loci/genes listed in Supplementary Table http://genome.ucsc.edu/) and variants were identified using the Ion Torrent pipeline .The exomes of the parents and affected children were sequenced on the Ion Proton platforms, using the whole exome AmpliSeq kit. Briefly, 12 separate Exome Primer Pools, AmpliSeq HiFi mix  and 100 ng DNA from each sample were used in the amplification step for 10 cycles. The resultant PCR products were then pooled in preparation for primer digestion using FuPa reagent . This was followed by a ligation step using Ion P1 and Ion Xpress Barcode adapters. The libraries were then purified and quantified using qPCR and the Ion Library Quantification Kit  prior to emulsion on an Ion OneTouch System. The templated Ion Sphere particles were enriched using Ion OneTouch ES  and processed for sequencing on the Ion Proton instrument . Approximately 15–17 Gb of DNA sequence was generated per sequencing run/sample. Reads were mapped to UCSC Human reference genome (hg19) . The analysis pipeline is a multi-step process . On the other hand, the variant was considered autosomal dominant only if it was transmitted to the proband from one heterozygous parent. Moreover, for a variant to be considered as X-linked, it had to occur on the X-chromosome in either a hemizygous  or a homozygous  state in the proband in reference to the parents.As part of a tertiary data analysis, each trio was further interrogated using four possible inheritance models; autosomal recessive, autosomal dominant, http://annovar.openbioinformatics.org) software was used to functionally annotate detected variants as previously described98. Briefly, the software was used to perform two types of functional annotations; gene-based and filter-based. The former one identifies the genomic region containing the variant (e.g exonic or intronic), and the latter assesses the frequency of the variant in widely used databases in addition to providing functional prediction scores from a number of tools.ANNOVAR  were checked for reported SNVs and CNVs in our list of validated genes or bands harboring these genes in case of CNV105. Finally, segregation analysis was carried out in families where DNA from unaffected family members (siblings) was available .The resulting model-specific set of variants was trimmed by omitting the ones not falling within coding exons or exon/intron boundaries, or non-functional. Then validation by Sanger sequencing was carried out on fresh DNA aliquots prior to assessing the frequency and predicted functional effect of the detected variants. The purpose of this was to avoid overlooking any interesting changes that may have been masked by variant calling/annotation errors. The list of validated variants was further trimmed by omitting those reported in dbSNP, or present in international databases  or in the local ethnically matching normal controls database (SHGP 2379 exomes) with a MAF ≥ 1%. Variant deleteriousness was predicted using four different web-based tools: Polyphen-2, SIFT, MutationTaster and CADD as describedet al. method, which is based on the Ajk statistics106. For this method we used the implementation in the VCFtools package (with option –relatedness 1). In the second method, we computed shared homozygosity by comparing the homozygous variants (MAF > 1%) between each pair of individuals and counting the number of overlapping variants. Relatedness was denoted as “Not confirmed” if the number of shared homozygous variants was less than 25 SD above the mean (123 ± 25). The third method estimates kinship as described by Manichaikul and colleagues107 using KING program (http://people.virginia.edu/~wc9c/KING/) and the implementation in VCFtools (option –relatedness2).Three different relationship inference algorithms were employed in this study to verify the self-reported pedigree information. The first is Yang https://analysis.ingenuity.com/pa/installer/select). The software identifies the diseases and biological functions that are significantly associated with our genes list. The resulting categories are ranked by p-values calculated using right-tailed Fisher’s exact test.In an attempt to gain insight into the biological processes that may potentially be affected by the variants identified herein, we analyzed our final list of genes with validated variants using the core analysis function of Ingenuity Pathway Analysis software (IPA)  into different categories including: “Neuronal function/Development”, “Mitochondrial function/Energy metabolism”, “Protein quality control”, “Gene regulation”, “Cell signaling cascade”, “Cell division or differentiation”, and “Immune responses” Table .Supplementary informationDataset1


Phosphorylation of the regulatory light chain (RLC) of myosin modulates cellular functions such as muscle contraction, mitosis, and cytokinesis. Phosphorylation defects are implicated in a number of diseases. Here we focus on striated muscle where changes in RLC phosphorylation relate to diseases such as hypertrophic cardiomyopathy and muscular dystrophy, or age-related changes. RLC phosphorylation in smooth muscle and non-muscle cells are covered briefly where relevant. There is much scientific interest in controlling the phosphorylation levels of RLC in vivo and in vitro in order to understand its physiological function in striated muscles. A summary of available and emerging in vivo and in vitro methods is presented. The physiological role of RLC phosphorylation and novel pathways are discussed to highlight the differences between muscle types and to gain insights into disease processes. RLC phosphorylation acts as a modulator and not as a switch in striated muscle  is a member of the EF-hand superfamily . The N-terminal domain is similar in structure to calmodulin. RLC is non-covalently bound to the C-terminus of myosin S1 subfragment  and type II (NCBI Accession # P02608), and one slow isoform , where fast and slow describe the physiological behavior of different muscle groups. These three isoforms are encoded by different genes in the human chromosome 16, but with a high degree of sequence homology Szczesna .In human heart, there are two different RLC isoforms expressed from separate genes, namely the atrial isoform (MYL2a) (NCBI Accession # M94547) encoded from a gene in chromosome 7 and the ventricular isoform (MYL2v) (NCBI Accession # P10916) encoded from a gene in chromosome 12, with the ventricular ones expressed in both ventricular myocardium and slow twitch skeletal muscle (MYL2f)   residues, Ser14 and Ser15 Fig. , by a skIn striated cardiac muscle from mouse or rat, RLC exists in three states: unphosphorylated, singly phosphorylated at serine 14 or 15, and doubly phosphorylated at serine 14 and 15. These sites are mostly phosphorylated by a cardiac-specific MLCK (cMLCK), and partly by zipper-interacting kinases (ZIP Kinase). While mouse and rat possesses phosphorylatable sites at both Ser14 and Ser15, human cardiac RLC (cRLC) has only one single phosphorylatable site at Ser15 Fig.  Lyon et. Interes2+-phos-tag gel electrophoresis technology for detecting phosphorylated variants of proteins, Aguilar et al. showed three differentially charged species of RLC in cultured uterine smooth muscle myocyte: unphosphorylated, singly and doubly phosphorylated  phosphorylates Ser1, Ser2, and Thr9 of smRLC, which reverses the increase in ATPase activity associated with smMLCK phosphorylation at Ser19  in the presence of calcium and calmodulin  of muscle  mouse, the extensor digitorum longus (EDL) muscle exhibits no post-tetanic force potentiation after potentiating stimuli compared to that in wild-type control mice  mutations known in human cardiac RLC are found in the region of the Ser15 phosphorylation site, affect RLC phosphorylation and have a physiological effect  and troponin I (TnI), are also affected by in vivo mutations of cRLC phosphorylation sites  biopsy samples from patients either with LV hypertrophy and preserved systolic function or with LV dilation and reduced ejection fraction  by crossing cMLCK/neo+ to each other in order to assess myosin RLC phosphorylation and cardiac performance in vivo , smMLCK expression is tissue-specifically abolished  or glutamic acid (E)], which carries a negative charge that mimics the charged state of a phosphorylated residue. Based on the known cRLC crystal structure and on molecular dynamics (MD) simulations, the in vivo phosphorylatable site of Ser15 is proximal to the D166V  mutation site of cRLC known to cause familial hypertrophic cardiomyopathy (FHC). Muthu et al. created a S15D (phosphorylated Ser15) pseudophosphorylated mutation site in the cRLC protein carrying the D166V mutation  activity , which induces cardiac hypertrophy in wild-type mice and is associated with an increase in MYL2v phosphorylation in mice that lack cMLCK, by activating the ZIP Kinase  plays an important role in regulating vascular tone as well as in maintaining systemic blood pressure. Volatile anesthetics may inhibit AngII-induced vascular contraction via inhibiting RLC phosphorylation  and Michaelis constant (Km)  whereas smMLCK proceeds by an ordered sequential mechanism in which Mg-ATP binds first, followed by the RLC substrate  is expressed in most tissue including smooth muscle, skeletal muscle, and cardiac muscle. smMLCK catalyzes CaDrosophila  and incubated with 0.1–0.5 µM RLC for 0.5–2 h at 25–37 °C or at 4 °C overnight. This method yields almost 100 % cRLC phosphorylation, i.e., the cRLC species are phosphorylated at Ser15 position with almost 100 % efficiency  dissolved in buffer . ATPγS was used because thiophosphorylated proteins are relatively resistant to dephosphorylation by contaminating or endogenous protein phosphatases in the muscle tissue samples  are also used to reduce RLC phosphorylation in vitro  and protein kinase C (PKC). Rho-kinase is also involved in RLC phosphorylation in striated muscle. For example, adult rat ventricular myocytes were treated with the Rho-kinase inhibitor Y-27632 at a concentration of 10 µM so that it does not phosphorylate the myosin-binding phosphatase targeting subunit (MYPT), the catalytic subunit of protein phosphatase 1 (PP1)  and P34 kinase also affect RLC phosphorylation, but most reports refer to smooth muscle and non-muscle cells  have several disadvantages, especially when targeting a specific kinase to modulate RLC or other sarcomeric protein phosphorylation. Since the ATP-binding sites of kinases are highly conserved, it is difficult to target kinases specifically using pharmacological inhibitors. On the other hand, genetic approaches to deactivate kinases are very specific but take time to be effective and levels of inactivation are difficult to control. Small molecule pharmacological inhibitors inactivate kinases rapidly and in a dose-dependent manner. Moreover, interpretation of results using genetic or pharmacological techniques is complicated by functional redundancy of kinases and the overlapping substrates of multiple kinases. To overcome this, Kevan Shokat and his colleagues  as substrate. Using SDS-PAGE and autoradiography, RLC was found to be the direct substrate of ZIP kinase in situ. Thus, this approach manipulates RLC phosphorylation in different tissue systems by selectively inhibiting kinases.This chemical genetic approach has been exploited to identify RLC as a direct substrate for phosphorylation by ZIP kinase to regulate smooth muscle contraction  pathway  gel analysis is most common to detect phosphovariants of proteins according to their isoelectric point by isoelectric focusing and subsequently by molecular weight via SDS-PAGE (O’Farrell DE gel an®) selectively stain phosphoproteins in acrylamide gels, without the need for blotting or phosphoprotein-specific antibodies and Western-blot analysis  as it can be conjugated with protease digestion and tandem mass spectrometry   and TnI in cardiac muscle . There are several molecular players that control this dynamic process. Along with regulation via MLCK activity (reviewed above), MLCP also keeps the activity balance  was identified in smooth muscle  studies may be used to observe the dynamics of cross-bridge interactions in different phosphorylation states of RLC. These studies may focus on the structure of the cross-bridges, but also in the inter-filament spacing and myofibrillar organization. Pseudophosphorylation may provide stable systems to aid structural studies and molecular dynamic simulations. Results will assist the elaboration of models of contraction in which the contractile performance and efficiency of muscle is related to disease states encountered in hypertrophic or dilated cardiomyopathies.RLC phosphorylation regulates muscle contraction and other cellular functions, with important consequences for muscle behavior in health and disease. RLC phosphorylation plays a part in muscle disorders and may be a future therapeutic target. This review summarizes the structure and function of RLC and gives an overview of the methods available for manipulating and testing the effect of RLC phosphorylation in muscle tissue, focusing on striated muscle. We also compare the advantages and disadvantages of in vivo and in vitro methods and suggest their potential applications.


Even though branchial cleft cysts are currently accepted as a congenital anomaly, there is often a long delay until clinical presentation; branchial cleft cysts classically appear in the second to fourth decade of life. Our observation of their occurrence in three pregnant women encouraged us to contemplate a possible hormonal influence.α) in paraffin-embedded tissue specimens of 16 patients with a diagnosis of branchial cleft cyst, with three of them being pregnant. Immunohistological analysis was performed for the evaluation of the estrogen receptor alpha  were found. Expression of ER The fact that the estrogen receptor was expressed only in pregnant women, in contrast to 13 investigated cases, may suggest that the high level of estrogen in pregnancy is a possible explanation for the spontaneous growth of branchial cleft cysts. A broad spectrum of pathologies manifest themselves by swelling in the neck as an early symptom. Beyond frequent reasons such as abscesses or neoplasms, a branchial anomaly is causative in only 0.2% of all cases, with an overall occurrence rate of 1 : 3500–4000 [α) expression pattern in these three cases and compared them to branchial cleft cysts resected from nonpregnant women and men of different ages.Our clinical observation of the occurrence of branchial cleft cysts in three pregnant women, presenting in our department within two years, gave rise to the question of whether estrogen, which is elevated during pregnancy, is able to induce the growth of these branchial cleft cysts. Therefore, we performed an immunohistological study analyzing the growth fraction (Ki67/Mib-1) and the estrogen receptor alpha .For the purpose of this study, we investigated paraffin-embedded tissue specimens from 16 patients with a diagnosis of branchial cleft cysts. Seven patients were female, with three of them being pregnant  along with three nonpregnant  women; the remaining female subject was a 4-month-old child. The other nine patients were males of different ages (ranging from 8 to 82 years). Histological analysis confirmed the diagnosis of a branchial cleft cyst in all of these cases Figures .Within a period of 24 months, the three pregnant women presented consecutively with a growing, firm elastic mass in the anterior triangle of the neck . By MRI,In all cases, the cysts were extirpated under general anesthesia without any complications Figures . The lymImmunohistochemical analysis was performed on 4-mm sections obtained from formalin-fixed paraffin-embedded material. The primary antibodies used for the evaluation were antiestrogen receptor alpha (clone SP1) from Ventana Medical Systems  provided as a ready-to-use antibody and Ki-67 clone Mib-1 obtained from Dako  applied at a concentration of 1 : 100. The sections were dewaxed and subjected to an antigen retrieval protocol within a BenchMark Ultra instrument  followed by incubation with primary antibodies. Bound antibodies were visualized using the streptavidin–biotin–peroxidase method and diaminobenzidine as the chromogen . For positive controls, tissue microarray sections from breast carcinoma cases were used. As a negative control we incubated tissue sections with commercially obtained IgG-antibodies of the same subclass as the monoclonal antibodies used for Ki-67/MiB1 and ERalpha.α in nuclei of a portion of the epithelium  showed a lower proliferative activity than those detected during pregnancy.Both evaluable cases of pregnant women showed weak nuclear expression of the ERα and estrogen receptor beta (ERβ), which act as hormone-inducible transcription factors [β-estradiol circulates in the blood; thereby, this free steroid diffuses in and out of all cells [α or ERβ at the cell surface, the receptors dimerize and bind with high affinity in the regulatory regions of their target genes [It is well known that estrogen plays an important role in cell proliferation and growth within and outside the reproductive system . These e factors  and belo factors . During ll cells . When eset genes , 12.β (E2) controls epithelial proliferation via cross-talk between epithelial-stromal cell layers [β-estradiol have been described [Many studies regarding the regulatory effects of estrogen in different organs outside the reproductive system, especially in the liver, brain, and retinal endothelial cells , 13, 14,l layers , althougl layers . In partescribed . Estrogeescribed . Spencerescribed . Moreoveescribed . These dα in various cell types such as liver cells [α-expression within epithelial cells in branchial cleft cysts. Consequently, the observations presented in this study might be a nonspecific phenomenon in pregnant women. However, the fact that in 3 out of 7 female patients the branchial cleft cyst became clinical apparent during pregnancy supports the hypothesis that estrogen might have influenced the growth of the cysts through ERα. The exact reason for the different receptor expression between pregnant and nonpregnant patients cannot be concluded from this study. The evaluation of more patients might lead to further information.As described, estrogen can induce the expression of ERer cells  and breaer cells . Based oThe effect of pregnancy on tumors remains a major concern today. In the current literature, there are a few reports mentioning an association between steroid hormones/ER and soft tissue tumors  , 23. Theα expression in the epithelial lining of branchial cleft cysts in only two cases arising in pregnant females, in contrast to all other cysts arising in males and nonpregnant females, this is nevertheless the first study to postulate a tissue-based explanation for the up to now unexplained growth of branchial cleft cysts in pregnant women.Although we could demonstrate ER


The presence and relative stability of extracellular RNAs (exRNAs) in biofluids has led to an emerging recognition of their promise as ‘liquid biopsies’ for diseases. Most prior studies on discovery of exRNAs as disease-specific biomarkers have focused on microRNAs (miRNAs) using technologies such as qRT-PCR and microarrays. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species). Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. There is reasonable correlation of sets of conserved miRNAs across biological replicates, and even across other data sets obtained at different investigative sites. Conversely, the detection of less abundant miRNAs is far more dependent on the exact methodology of RNA isolation and profiling. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms. More recently, the application of next generation sequencing to exRNA discovery has led to the recognition that various other species of RNAs are also present in biofluids [Extracellular RNAs (exRNA) have recently been identified as novel biomarkers and potential cell-cell communicators in plasma and other body fluids. To date, the most abundantly studied class of these exRNAs are microRNAs (miRNA), which have been implicated in a wide range of diseases including heart failure , 2, canciofluids , 11. Whiiofluids , and as While exRNA offers an exciting new branch of functional biomarker research, the field is still in its infancy and faces numerous technical challenges. RNA concentrations in biofluids are significantly lower than in tissue and standard methodologies developed for tissue RNA extraction and quantification are not always appropriate. Secondly, the RNAs may be present in different compartments, each of which may have different susceptibilities and vulnerabilities to RNA isolation techniques, thereby creating an unintended bias in RNA profiling based on the exact isolation method used , 14. FurCurrent technologies available for the quantification of exRNA include qRT-PCR, RNA microarray, and RNA sequencing (RNAseq). While qRT-PCR and RNA microarrays utilize specific primers or probes, and as such can only detect known RNA sequences, high through-put RNAseq has the ability to detect novel transcripts across a broad dynamic range and offers a potentially sensitive means to characterize and quantify exRNA. However, in addition to the biases introduced by the technique , 15, theAll experiments were conducted with approval of the Institutional Review Board of Beth Israel Deaconess Medical Center and written consent of the subjects. Blood was obtained from 4 healthy adult volunteers  between the ages of 20–40 for all experiments except the long archived ribodepleted vs non-ribodepleted samples. These samples were obtained from a registry of post-myocardial infarction patients with collected plasma for assay of novel biomarkers (PROSPECT-CMR). These patients  were between the ages of 49–70, where Pt 1 and Pt 3 are females and Pt 2 is male.2EDTA-containing tubes. Blood was immediately processed by centrifugation at 1,000 x g for 10 min at room temperature to separate plasma. The isolated plasma was immediately frozen at -80°C in 1 mL aliquots until use. Samples were frozen for approximately 1 week prior to extraction, except where noted. Plasma was thawed at room temperature and centrifuged at 2,000 x g prior to RNA extraction to remove any remaining platelet or debris contamination.40 mL of blood was obtained by venipuncture using a 21G needle and collected in KRNA was extracted from 1 mL of plasma using the miRCURY RNA Isolation Kit for Biofluids (Exiqon) according to manufacturer’s protocol except for noted modifications. 1 μL of 20 mg/mL glycogen (Roche) was added to plasma prior to RNA extraction. All samples were treated with T4 polynucleotide kinase (New England Biolabs) to facilitate 5’ hydroxyl terminus phosphate labelling and allow greater binding of adaptors during library preparation, and RNA sample volume was reduced to ≤10 μL in a standard lyophilizer at room temperature. RNA concentration was determined by Quant-iT RiboGreen RNA Assay Kit.Prior to RNA extraction, a subset of plasma samples were treated with Proteinase K  for 30 min at 50°C. Samples were treated in one of three groups: 1) no PK treatment; 2) PK treatment prior to addition of guanidinium thiocyanate (GITC) containing lysis buffer (from miRCURY RNA Isolation Kit); 3) PK treatment following the addition of GITC-containing lysis buffer. Where indicated, ribodepletion was performed using the Ribo-Zero Magnetic Gold kit (Epicentre) according to manufacturer’s protocol. A schematic summarizing plasma sample treatments is shown in For samples that were analyzed by digital droplet PCR (ddPCR), ethanol precipitation was performed to remove any excess SDS solution that could inhibit the PCR reaction. Briefly, 0.3 M NaCl, and 3x volumes of 100% ethanol were added to the solution and the RNA precipitated at -80°C overnight. Samples were then pelleted by centrifugation, washed with 80% ethanol, and re-suspended in 10 μL RNAse-free water.Each sequencing library was constructed from 2 ng of isolated and treated plasma RNA. All libraries were uniquely bar-coded with index primer for multiplexing into sequencing lanes. The small RNA libraries were prepared and amplified using the NEBNext small RNA Library Prep Set  following manufacturer instruction. The amplified libraries were resolved on a 10% Novex TBE gel (Life technologies) for size selection and the 140 to 160 nucleotide bands that correspond to adapter-ligated constructs derived from the 21 to 40 nucleotide RNA fragments were excise and recovered in DNA elution buffer. The average size distribution of each library was determined using Agilent Bioanalyzer with High Sensitivity Chip Kit  and quantified on ABI 7900HT Fast RT-PCR instrument using the KAPA Library Quantification kit according to the manufacture’s protocol . Each library was adjusted to final concentration of 2 nM, pooled, and sequenced on an Illumina HiSeq 2000 or MiSeq sequencer for single read 50 cycles at the Center for Cancer Computational Biology at Dana-Farber Cancer Institute.http://hannonlab.cshl.edu/fastx_toolkit). The processed sequences were filtered for small RNAs greater than 16 nucleotides in length. The sequences were then aligned, quantified and annotated using sRNABench 1.0 pipeline [http://exrna.org/resources/data/) as part of Extracellular RNA Communication Consortium (ERCC).The BCL files were de-multiplexed using CASAVA v1.82, and the adaptor sequences within the read sequences were trimmed by FastX-Toolkit  according to manufacturer’s protocol. cDNA was diluted 1:10 and loaded into PCR reactions with EvaGreen supermix (Bio-Rad) and pre-designed primers for miR-30d, miR-150, and miR-122 (Exiqon) according to manufacturer’s protocol (Exiqon). Droplet formation was carried out using a QX200 droplet generator and droplets were transferred to a 96-well plate for PCR. No template reactions were used as a negative control. Endpoint amplification was conducted with the following steps: 95°C for 5 min, 40 cycles of 95°C for 30 s, 58°C for 30 s, 4°C for 5 min and 90°C for 5 min at a ramp-rate of 2.5°C/s for all steps. Droplets were then read with the QX200 droplet detector (Bio-Rad) and analysis conducted with Quantsoft 1.7. Results are presented as expression relative to the no PK treatment counts.a) no PK, b) PK treatment before GITC (Pre-GITC), and c) PK treatment in GITC (PK in GITC and PK in GITC #2) that was drawn from a single subject. The no PK, PK pre-GITC, and PK in GITC samples were from the same blood draw, whereas the PK in GITC #2 was from a separate draw. Total RNA yield from these samples  was a) 22.1 ± 1.3 ng, b) 23.0 ± 3.2 ng, and c) 67.0 ± 6.0 ng (mean ± SEM). Libraries were constructed using NEBNext small RNA Library Prep Set and run on the Illumina HiSeq2000 platform. The number of initial reads post-filtering varies from 5.5 to 8.4 million for each sample, with more than 50% of the reads being ribosomal RNA (rRNA) (To determine the impact of proteinase K (PK) treatment at different stages of RNA extraction affecting miRNA detection, we extracted RNA from plasma treated with A (rRNA) . The rRNDespite the large variation in the total number of miRNA reads, all three samples yielded more than 346 mature miRNA with PK-in-GITC sample yielding the most diverse result (n = 428). Overall we identified reads mapping to 593 mature miRNA across samples with detectable counts count>0, , but onlLastly we checked if commonly observed miRNA species were expressed at higher levels and thus more resistant to technical variation. miRNA that were observed in all samples showed significantly higher expression compared to those observed in two or less samples . Similarly, miRNAs uniquely observed in only one sample generally have lower expression, particularly among those uniquely identified from the PK in GITC sample. We repeated PK-in-GITC treatment on a different plasma sample (PK-in-GITC #2) derived from the same subject, and observed similar level of percentage mapped reads 31.3%, . Taken tTo further validate these findings we performed ddPCR on repeated samples of no PK vs PK in GITC from the original single healthy donor. From the RNAseq results, we selected a high abundant (miR-122), mid-abundant (miR-30d), and low abundant miRNA (miR-150) species to measure . InteresDue to the high percentage of rRNA reads observed, the effect of ribodepletion on sample reads was tested. Plasma samples from the same healthy individual, frozen for either 1 week (fresh) or 1 year (archived) were extracted with PK pre-treatment and ribodepletion was performed. As expected, ribodepleted fresh samples showed decreases in the percentage of rRNA reads, ranging from 15 to 25%, while the percentages of miRNA reads and detectable mature miRNA species both showed increases  when comTo further validate this observation, we sequenced both ribodepleted and non-treated archived plasma samples derived from three cardiovascular patients (subjects Pt1 to Pt3) that were archived for 3–4 years. The result concurred with previous observation. Ribodepleted archived samples consistently yield dramatically lower percentage of miRNA mapping reads and lower number of miRNA species compare to non-treated samples . ParadoxTo assess technical reproducibility of miRNA profiling, we examined duplicated miRNA profiles derived from the plasma of three healthy subjects between the ages of 20 to 40 . Plasma samples collected from umbilical cords showed slightly weaker correlation. On the other hand, correlations between placenta and plasma data were generally weaker  despite showing similar high miRNA expression level among the conserved miRNAs as other sample types. This data indicates that there may exist conserved miRNA expression profiles that are specific to plasma.Next we evaluated if these conserved miRNA expression patterns were consistent between the plasma samples from the two datasets. Correlations across datasets for plasma samples were strong  for plasWe examined the number of reads that were mapped to other class of small RNAs in addition to rRNA and miRNA. Overall, among the total 558,000 reads of small RNA sequences generated from the three healthy subjects, there were 67% miRNA, 23% tRNA, 5% small nucleolar RNA (snoRNA), and 5% piwi-interacting RNA (piRNA) reads mapped . DespiteWe began this study with the intention to establish a robust small RNA extraction procedure for peripheral blood plasma miRNA quantification that can be reliably utilized for biomarker discovery from clinical samples. While settling on a single kit based on other studies, our goal was to examine the effects of modifications of RNA extraction procedures for assessing plasma miRNA expression profiles using NGS as our quantification assay. The experiment was designed to select the optimal RNA extraction procedure on plasma samples derived from a single healthy individual, taking consideration of sample storage duration, and then extended to multiple individuals as well as published data to evaluate consistency.We first evaluated the impact of PK treatment, which has been reported to increase RNA yield , on extrOne of the most surprising outcomes from our sequencing data was the consistently high percentage of ribosomal RNA reads that occupies large proportion of sequence reads. This made it more challenging to achieve a high depth of coverage for profiling miRNA expression. We tested the ability of ribodepletion, which is standardly performed for RNAseq of tissues samples, to remove rRNA from extracted plasma RNA derived from fresh peripheral blood plasma. The result was initially highly encouraging with both substantial decrease in the percentage of rRNA and increase in the percentage of miRNA reads. However, the same plasma sample that were frozen for one year yielded poor sequencing results after ribodepletion, with no reduction in the percentage of rRNA and substantial decrease in the percentage of miRNA reads. As most clinical samples would have been archived for extended period of time, it is important to note that fresh and archived samples should be handled differently. The use of ribodepletion, while potentially beneficial in fresh samples, may not be suitable for the study of archived samples. Other techniques, such as blocking of highly abundant miRNAs for plasma RNA sequencing, have been described but not adapted to multiplexing and could offer another avenue of increasing the detection of less abundant species of exRNAs.Our comparisons of miRNA expression profiles showed strong correlation of miRNA species for both sample replicates and between samples from different healthy donors, supporting the reproducibility of NGS as a methodology for plasma miRNA quantification. In particular, despite the low RNA concentration within peripheral blood plasma that may introduce strong sampling bias, large numbers of abundantly expressed miRNA species were observed across samples that we examined in this study with similar profiles. We further investigated if these conserved plasma miRNA that we observed exhibit similar patterns from healthy individuals in published study and found that these profiles remain strongly correlated with our results despite differences in sample acquisition, RNA extraction, and sequence library preparation protocols. While this may suggest the presence of a stable landscape of extracellular miRNA in healthy plasma, further studies with larger cohorts are required to confirm this observation.As the scope of this study is limited to optimize only small aspects of exRNA extraction process from plasma for small RNAseq quantification, there remain arrays of challenges in generating reproducible and reliable data. For instance, T4 polynucleotide kinase treatment during sequencing library generation likely facilitated extracellular DNA nucleotide for sequencing  to resulS1 TablePlasma was treated with a) no proteinase K(No_PK); b) PK treatment before GITC (PK_pre_GITC); c) PK treatment in GITC (PK_in_GITC); d) Repeated PK treatment in GITC (PK_in_GITC_2)(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


Gas1 and Boc were used to generate Gas1; Boc compound mutants. The expression of key Hedgehog signaling family members was examined in detail during palatogenesis via radioactive in situ hybridization. Morphometric analysis involved computational quantification of BrdU-labeling and cell packing; whilst TUNEL staining was used to assay cell death. Ablation of Boc in a Gas1 mutant background leads to reduced Shh activity in the palatal shelves and an increase in the penetrance and severity of cleft palate, associated with failed elevation, increased proliferation and reduced cell death. Our findings suggest a dual requirement for Boc and Gas1 during early development of the palate, mediating cell cycle regulation during growth and subsequent fusion of the palatal shelves.Abnormal regulation of Sonic hedgehog (Shh) signaling has been described in a variety of human cancers and developmental anomalies, which highlights the essential role of this signaling molecule in cell cycle regulation and embryonic development. Gas1 and Boc are membrane co-receptors for Shh, which demonstrate overlapping domains of expression in the early face. This study aims to investigate potential interactions between these co-receptors during formation of the secondary palate. Mice with targeted mutation in  Development of the mammalian secondary palate is a complex process, which requires a coordinated network of molecular and cellular events to produce appropriate growth, elevation and fusion of the constituent palatal shelves –3. In huThe Hedgehog (Hh) family of signaling molecules have emerged as major contributors to the developmental process in a wide variety of organisms, coordinating cell proliferation, survival and differentiation in multiple tissues –13. DysfGAS1 was mapped to human chromosome 9q21.3-22.1 and established as a negative cell cycle regulator and tumor suppressor [Gas1 was established through immunoprecipitation assays demonstrating Gas1 as capable of binding Shh and reducing its action [in vivo studies have argued against these initial in vitro observations [Gas1 mutant mice have demonstrated malformations characteristic of Shh loss-of-function, including micropthalmia [Shh dosage in a Gas1 mutant background leads to even more severe developmental defects [Gas1 is a positive component of the Shh signaling pathway [Boc was identified via screening of a human fetal brain cDNA library using a rat Cdon cDNA probe [BOC localizes to the plus strand of human chromosome 3q13.2 [Boc and Shh signaling [Gas1 is a N-glycosylated glycosylphosphatidyl inositol (GPI)-linked plasma membrane protein originally isolated via differential screening of fibroblasts maintained under growth arrest , 37. Subppressor . The firs action . Howeverrvations , 40, 41.pthalmia , HPE [27pthalmia , axon gu pathway , 40. BocNA probe . BiochemNA probe , 44. BOCe 3q13.2 . A studyignaling . Boc wasignaling . Moreoveignaling . Interesignaling . Recentlignaling . Taken tignaling .Shh transcriptional activity is detected in epithelium of the developing PS [Pax9 from mesenchyme of the PS has been shown to indirectly regulate Shh expression in the adjacent epithelium and downregulate key targets in the mesenchyme , placing Pax9 upstream of this complex gene network [−/−Gas1 mice also demonstrate cleft of the secondary palate (CP) with 50% penetrance, which is associated with reduced Shh signal transduction [Shh in the PS epithelium under control of a Keratin-14 promotor (K14-Shh) demonstrate reduced cell death in the MEE, which prevents PS fusion [oping PS , 49 and oping PS . Shh is oping PS . Shh is oping PS , 53. Mor network , 55. Gassduction . We haveS fusion . CollectGas1 and Boc has highlighted their importance in human diseases, including cancer [Gas1 and Boc during cell cycle regulation in the developing palate. Significantly, ablation of Boc in a Gas1 mutant background led to reduced Shh activity in the PS and increased severity of the CP phenotype. This was associated with failed PS elevation, increased mesenchymal proliferation and reduced epithelial cell death. Our findings suggest a dual requirement for Boc and Gas1 during early palatogenesis, mediating cell proliferation during growth and cell survival during subsequent PS fusion.There are currently over one thousand identified loci associated with orofacial clefting , but onlg cancer , 62–64. Shh transcriptional activity was observed in the developing rugae of the PS oral epithelium between E12.5-14.5 . Table Post hoc pairwise comparisons demonstrated that the only non-statistical significant result was the CPI difference between −/−; Boc−/−Gas1 and +/−; Boc+/−Gas1 PS (p = 0.636)  ; whereas −/−Boc PS showed the lowest CPI amongst genotypes  if the cell density is constant, any differences in the PIPUA denotes a true change in proliferation as a result of mutation in Gas1 and Boc or genetic interaction. Analysis of the PIPUA revealed a statistical significant difference among the four genotypes (p < 0.001). Table Post hoc pairwise comparisons among groups revealed a statistical significant difference between PIPUA amongst all genotypes, except for −/−; Boc−/−Gas1 versus −/−; Boc+/−Gas1 , whilst −/−; Boc+/−Gas1 and −/−; Boc−/−Gas1 PS also demonstrated a higher PIPUA compared to control, but to a lesser extent  Figure . In facts Figure , Table 2S p = 0.66  taking into account the interdependent relationship of the two quantities [−/−Boc PS presented a decreased cell density (CPI) and increased proliferation (PIPUA) in comparison to control . These seemingly conflicting results can be explained by two possible mechanisms: (1) either an increased compensatory apoptosis, resulting in a net reduction in cell number or (2) an increase in the average distance between cells as a result of an increase in overall tissue size. The former hypothesis can be excluded following the apoptosis analysis, which demonstrated cell death present primarily in the epithelium at levels similar to those observed in +/−; Boc+/−Gas PS  PIPUA observed in the −/−; Boc−/−Gas PS. Although both genes seem to be negative regulators of proliferation in this developmental context, it is highly suggestive that additional regulators play a role in this network. Moreover, higher CPI and PIPUA are not necessarily an indication of aberrant palatogenesis, as observed in −/−Boc embryos. Therefore, it is reasonable to speculate that the HPE midline facial anomalies present in −/−; Boc−/−Gas1 [Whole population cell analysis of the PS epithelium has highlighted the importance of considering factors other than cell proliferation in isolation when evaluating tissue growth . Here weantities , 65, 69.S Figure . Similarpoptosis . As no oferation , 71. Intogenesis , 72. Gasogenesis  and has ogenesis  and glioogenesis . In Gas−; Boc−/−  could plGas1; Boc mutant PS phenotype. Histological analysis demonstrated that the −/−; Boc−/−Gas1 PS size are similar to that of +/−; Boc+/−Gas. Therefore, in order to further understand how the observed differences in proliferation contribute to the CP phenotype, a direct measure of the overall midfacial region of −/−; Boc−/−Gas1 mice would be required. This may prove challenging to perform in plane section, because no account would be taken of cellular movements and rearrangements that might be taking place in the z- dimension [−/−; Boc−/−Gas1 mice) that correlates with reduced transcriptional activity of Shh signaling readouts. Furthermore, deletion of Gas1 leads to reduced apoptosis in the PS. Although the CP phenotype in mice lacking Gas1 [Gas1; Boc mutants is associated with PS that fail to elevate above the tongue; it is unlikely that the PS would fuse, as demonstrated by transgenic mice over-expressing Shh in the oral epithelium [We have excluded tissue packing changes as a potential cellular mechanism underlying the imension , 69. Altimension . Similarimension . The resing Gas1  or in coithelium .Boc in a Gas1 mutant background leads to reduced transduction of Shh signaling. Morphometric analysis revealed that the more severe clefting phenotype observed in these mice was associated with higher proliferation levels and reduced apoptosis. Additional mRNA expression analysis of known mediators of palatal development may help to further define a gene network in developing palate. While systems approaches are important to elucidate the vast molecular network regulating complex developmental processes such as palatogenesis, understanding the role of individual genes implicated in cell regulation is also highly valuable. This study has directly addressed the role of two key Hh signaling components and their dual requirement for orchestrating palatogenesis. Similar studies addressing the roles of other key Hh components should eventually lead to a more complete picture of the genetic basis of midline development and how it relates to human syndromic disorders.The results from this study further highlight the importance of Shh signaling in coordinating the process of palatogenesis. Hh family members are expressed at key stages during palate development , 56, 61.−/−Gas1 mice were generated and maintained in a 129sv/C57BL6 mixed background and genotyped as previously described [−/−Boc mice were generated and maintained in a CD1/129sv mixed background and genotyped as previously described [+/−Gas1 mice were crossed with +/−Boc mice, to generate −/−Gas1; −/−Boc compound mutants in a mixed background (129sv/C57BL/6/CD1). Timed-matings were set up such that noon of the day on which vaginal plugs were detected was considered as embryonic day (E) 0.5.All mice were housed and all experiments conducted in compliance with the approved protocols at King's College London, UK and the Carnegie Institution of Washington, USA. escribed . Boc−/− escribed . Gas1+/−For histological analysis, embryos were fixed in 4% paraformaldehyde (PFA) at 4°C, dehydrated through a graded ethanol series, embedded in paraffin wax, sectioned at 7 mm and stained with haematoxylin and eosin (H&E).in situ hybridization was carried out as previously described [Radioactive section escribed . Dark-fiA CPI was generated by dividing the total number of cells by the area of the region of interest. Assays for cell proliferation were carried out using a Zymed Bromodeoxyuridine (BrdU) Labeling and Detection Kit (Invitrogen), according to the manufacturer instructions. Mouse embryos were labeled with BrdU via intra-peritoneal injection into pregnant females (5 mg/100 g body weight) 2 hours prior to sacrifice. Slides were photographed using a Zeiss Axioscop microscope (Germany).−3 and PIPUA was multiplied by 10−9. The graph illustrating the PIPUA is in a logarithmic scale to facilitate visualization of the data.The imaging software package FIJI  was used® Plus Fluorescein In Situ Apoptosis Detection Kit  according to the manufacturer's instructions. Slides were photographed using a Zeiss Axioscop microscope (Germany).Immunohistochemical detection of apoptotic cell death was carried out on histological sections using Terminal deoxynucleotidyl transferase-mediated deoxyUridine triPhosphate Nick End Labeling (TUNEL). TUNEL was carried out using an APOPTagp < 0.001 from the Shapiro-Wilk test for both), the median and interquartile range (IQR) are reported as descriptive statistics. The Kruskal-Wallis test was used to test for differences in CPI and PIPUA among the four groups. Differences among the four groups were identified by calculating coefficients and the corresponding 95% confidence intervals (95% CI) through generalized linear regression models, only if the null hypothesis was rejected with the Kruskal-Wallis test, so as to reduce the risk of increased Type II error. According to inspection of the histograms and to model fit, a Poisson distribution was adopted for the models with calculation of robust standard errors to control for mild violation of underlying assumptions [post hoc pairwise comparisons among groups were performed only in case of a statistically significant Kruskal-Wallis test and these were of explorative nature, no P-value correction was applied. All statistical analyses were conducted with a 2-sided α of 5% in Stata version 12  with the macros swilk, kwallis, and glm.The assumption of normality for each variable was checked with with the Shapiro-Wilk test . The assumptions . As post