Depression is one of the commonest co-existing medical conditions among patients with diabetes mellitus (DM). A bidirectional relationship between depression and DM exists, complicating glycaemic control leading to an increase in diabetic complications. There is a dearth of information regarding the prevalence of depression and associated factors among patients with DM in Botswana.This study aimed to determine the prevalence of depression and associated factors among patients with DM. The study also assessed the association between depression and glycaemic control.A tertiary diabetic referral clinic in Gaborone, Botswana.A sample of 260 randomly selected patients with DM was recruited in this cross-sectional study. Socio-demographic and clinical characteristics of the patients were collected using a case report form. Depression was evaluated using the Patient Health Questionnaire (PHQ)-9 scale. Multivariate regression analysis was used to determine factors significantly associated with depression.p-value = 0.004), three or more diabetes-related hospitalisations  and inversely associated with systolic blood pressure (SBP) ≥ 140 mmHg .The mean age (standard deviation [s.d.]) of study participants was 58.4 (11.8) years, and the majority, 160/260 (61.5%), were females. The prevalence of depression was 30.4% and significantly associated with female sex (adjusted odds ratio [AOR] = 5.529, Depression is a common problem among patients with DM in our setting. Routine screening of depression in diabetic patients to enable early detection and treatment is recommended. One important factor is a low socioeconomic status that increases the odds for type 2 diabetes16 but also appears to be a cause for depression.17 The other common relationship between DM and depression is poor sleep, lack of physical exercises and poor adherence to eating habits or self-care activities.18 A key candidate for a common pathway could be the activation and disturbance of the stress system. Chronic stress activates the hypothalamus–pituitary–adrenal axis (HPA-axis) and the sympathetic nervous system (SNS), increasing the production of cortisol in the adrenal cortex and the production of adrenaline and noradrenaline in the adrenal medulla.19 Chronic hypercortisolemia and prolonged SNS activation promote insulin resistance and visceral obesity and lead to metabolic syndrome and type 2 DM.20 In contrast, chronic stress has behavioural consequences: noradrenaline, cortisol and other hormones activate the fear system determining anxiety and anorexia; the same mediators cause suppression of the reward system, which produces depression and cravings for food, other substances or stress.20 Moreover, chronic stress induces immune dysfunction directly or through the HPA axis or SNS, increasing the production of inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumour necrosis alpha , which in high amounts induce insulin resistance and ultimately type 2 DM.22Recent studies showed that there are not any common genetic factors to account for the positive association between depression and type 1 or 2 DM.24 These include female gender, older age, lower levels of education,25 being single or unmarried26 and high body mass index (BMI).23 Other behavioural and clinical factors associated with increased risk of depression among patients with DM include social isolation, monthly household income, starting antidiabetic drugs at a young age27 and presence of long-term DM complication such as nephropathy.28Previous studies in different parts of the world have explored factors associated with depression among patients with DM in outpatient settings and identified several demographic, behavioural, and clinical factors that predispose diabetic patients to depression.29 There is limited data on the prevalence of comorbid depression and diabetes in SSA. A meta-analysis of nine studies with 2944 participants in Ethiopia revealed a prevalence of depression among DM patients of 39.73%.30 Studies with fewer participants included cross-sectional studies in Nigeria,31 Tanzania,32 Malawi33 and a Kenyan prospective study34 with the prevalence of 30%, 30%, 18% and 20.9%, respectively. In contrast, two studies conducted in South Africa yielded higher prevalence rates of 46% and 46.6%, respectively.36 The literature search did not reveal any publication on the association between diabetes and depression in Botswana. The study aimed to establish the prevalence of depression and its associated factors among diabetic patients attending a tertiary clinic in Gaborone, Botswana. It also compared the psychometric properties of the two depression screening tools: Patient Health Questionnaire (PHQ)-9 and Beck’s Depression Inventory (BDI)-II, among patients with DM in our setting.Evidence from multiple systematic reviews and randomised controlled studies involving large sample size of patients has shown that recognising depression and providing psychological plus pharmacological interventions results in clinically significant improvements in both depression symptoms and glycaemic control.A cross-sectional study was conducted at a tertiary diabetic clinic between 10 June 2019 and 30 August 2019. The clinic is situated in Gaborone, the capital city of Botswana and offers several services including consultation, dietician services, eye examinations and pharmacy. It acts as a referral clinic for patients with diabetic complications and poor glycaemic control from local clinics within Gaborone and neighbouring towns in Southern, Central and Eastern Botswana. The choice of this clinic makes it appropriate for this study given that it serves as a main tertiary clinic for diabetes in Botswana, and findings are likely to represent the population of DM that is difficult to treat in Botswana.We recruited patients who were 21 years of age or older attending the diabetic clinic during the study period and having had the diagnosis of DM for at least 3 months and who consented to participate in the study. Patients with underlying severe mental illness such as schizophrenia or inability to comprehend the explained content of the study that made them unable to consent were excluded.37 The prevalence (P) of depression among diabetic patients of 20% from a study in Jordan38 that used the PHQ-9 was utilised to obtain our sample size. It should be observed that this was a Middle Eastern study; it would have been ideal to use prevalence from SSA studies; however, at the time of protocol development, there were no similar published studies in SSA using PHQ-9. We considered using the prevalence of 41% by the study of Udedi et al. in Malawi;39 however, this prevalence was obtained using Structured Clinical Interview for DSM-IV (SCID) for depression tool and not PHQ-9. We added 5% from the obtained sample size anticipating missing variables because of recall or missing results reaching 258, which was rounded to the final sample size of 260 participants. The order of recruitment was randomised on each day. Five pieces of papers of equal sizes and shapes were numbered 1–5 for patients to pick and identify the first patient to be recruited on each recruitment day. On each clinic day, about 60–70 patients attend for consultations. Our intention was to recruit at least seven patients per day; hence every eighth eligible patient was systematically recruited.A minimum sample size of 246 participants was required as ascertained using a formula for cross-sectional study.The study protocol was approved by the Ministry of Health and Wellness Research Unit and all relevant Institutional Review Boards in Gaborone, Botswana (IRB). All recruited participants signed written informed consent forms. Consent included assessing participants’ information from their outpatient charts and medical electronic system. It was made clear for each participant that there was no remuneration, and there was no physical harm to the participants. To ensure confidentiality, the interview was conducted in a secured consultation room with only the researcher and participant present. The recruited participants were assigned unique reference numbers for the study. There was also a pre-emptive plan to inform the attending physicians and urgently refer patients with moderately severe and severe depression (PHQ-9 ≥ 15) or active suicidal ideations to a psychiatric facility.One bilingual Motswana nurse translated the socio-demographic and clinical section of the case report form into the Setswana. This was followed by an evaluation of the translated tool by the first author. Two bilingual Batswana nurses then back translated the Setswana version into English independently and then met with the first author to arrive at the consensus translations.40; hence the Setswana version was adopted for this study. We did not have permission to translate the BDI-II inventory tool; hence the English version was used throughout.Patient Health Questionnaire-9 has been previously used in Botswana in Setswana languageData were collected by the first author with the assistance of a trained research assistant who was a paramedic with experience of working as an assistant in clinical research. The research assistant was trained on the details of the research prior to data collection initiation. Training entailed interpretation of socio-demographic, clinical and depression variables. Informed written consent was obtained from each patient before the interview and study procedures. The study was introduced to all the potential participants whilst awaiting consultations on each day of recruitment. This was followed by approaching each participant separately for eligibility and consent. Efforts were made to recruit participants whilst awaiting their turn for physician’s consultation; however, in few occasions, participants were recruited after physician’s consultation. Data were captured through face-to-face interviews using a case report form to obtain socio-demographic and clinical characteristics of the study participants. Socio-demographic variables collected included age, gender, level of education, marital status, household size and main source of household income, income per month and employment status. Clinical variables obtained included the following information: type of diabetes, duration of diabetes, type of antidiabetic drugs in use, number of diabetes-related hospitalisations, presence of documented complications in patients’ chart , number of documented complications, history of hypertension (yes/no), documented human immunodeficiency virus (HIV) status within past 1 year (positive/negative/unknown).41 and was documented to have type 1 DM in their charts. Diabetes-related hospitalisation was defined according to ICD 10 coded E10–E14 and referred to admission because of coma with/without ketoacidosis, hyperosmolar coma and hypoglycaemic coma, renal, ophthalmic, neurologic and peripheral circulatory complications.42 Hypertension was defined using Joint National Committee (JNC) 8 as systolic blood pressure (SBP) of ≥ 140 mm Hg and/or diastolic blood pressure (DBP) of ≥ 90 mm Hg43 in at least two prior visits or by current use of antihypertensive drugs. Depression was a binary measure similar to previous studies with PHQ-9 cut-offs of ≥ 10 indicating the presence of depression whilst < 10 was regarded as the absence of a diagnosis of depression.44 For BDI-II, cut-off of ≥ 20 referred to depression, whereas < 20 indicated no depression.45 Patient Health Questionnaire-9 was used to assess for the presence and severity of depression for all the recruited participants.Participants were categorised to have type 1 DM if they were diagnosed to have DM before the age of 30 years, and insulin was their first antidiabetic medication46 As a result, quick and inexpensive methods are available to screen people in primary and secondary care settings.47 Many short questionnaires have been used to screen for depression in the general population, but only a few have been evaluated adequately in people with DM.48There is no validated tool to screen for depression in Botswana, and a formal diagnosis of depression requires a validated interview, but this can be time consuming.45 the Centre for Epidemiologic Studies Depression Scale,49 the PHQ50 and the Hospital Anxiety and Depression Scale (HADS).51Some of the most well-validated depression screening questionnaires sharing psychosomatic properties for people with DM include the Beck Depression Inventory,Based on the fact that the PHQ-9 had not been validated in Botswana at the time of conducting the study, Beck’s Depression Inventory-II inventory was also administered for the last 100 patients. The two tools were used for convergent analysis, allowing exploration of psychometric properties of the tools.56 The authors paid for the cost of 100 BDI II English version forms purchased from Pearson.Patient Health Questionnaire-9 tool is free for use and does not require permission for copyright use. This tool has been validated internationally and in some sub-Saharan African countries such as Malawi, South Africa, Kenya, Zimbabwe and Mozambique.2 (m2) and categorised as underweight (< 18.5), normal weight (18.5–24.9), overweight (25–29.9) and obese (≥ 30). Body mass index categorisation was performed according to the World Health Organization (WHO).57 Blood pressure measurements were taken for each patient during registration. A second blood pressure measurement was carried out by the research assistant; the mean of the two blood pressures was used during analysis. Categorisation of blood pressure was performed according to JNC 8 guidelines for optimal blood pressure treatment goals in patients with diabetes as ≥ 90 and ≥ 140 mmHg for poorly controlled DBP and SBP, respectively. In contrast, < 90 and < 140 mmHg referred to good control of DBP and SBP, respectively.43Physical examination of patients included measuring the weight and height of each patient. Weight and height were routinely measured during registration using a stadiometer when the participant was on light clothing without shoes. Body mass index was calculated as a ratio of weight (kg) divided by height58 Glycosylated haemoglobin (HbA1c) (performed within 3 months of index visit) was obtained from the computer system (Integrated Patients Management System [IPMS]) or patients’ files. If the patient had no recent result, the HbA1c was ordered on the day of interview (the test was performed free of charge).Glycosylated haemoglobin (HbA1c) is a measure of average glucose control over the preceding 3 months. Treatment goals for participants with DM were categorised as follows: desirable (< 7%), suboptimal (7% – 9%) and poor (≥ 9%).59 Odds ratios (ORs) were used to test associations between a binary dependent variable (depression) using both PHQ-9 score and independent variables . Bivariate and multivariate logistic regression analyses were used for evaluating the factors associated with depression. Only variables that were significant at 10% level of significance in the bivariate logistic regression were used in the multivariate regression model.Data collected were entered and cleaned with Statistical Package of Social Sciences (SPSS) version 20 followed by analysis using the R version 3.6.1 and Stata 14.0. Frequencies were used to describe the demographic and clinical characteristics of the respondents. To determine the appropriate summary statistics, continuous distributions were checked for normality using the Shapiro–Wilk’s test. If the normality assumption was met, the mean and standard deviation (s.d.) were used to summarise the data, whereas the median and interquartile ranges (IQR) were used for variables that were not distributed normally. The Cronbach’s alpha statistic was computed to determine the internal consistency of PHQ-9 score and BDI II in terms of measuring depression, whereas convergence validity to obtain a correlation of two tools was computed by William’s test. Internal consistency equal or above 0.7 was considered acceptable.p-value of less than 0.05 was considered to be statistically significant.The stepwise model selection technique was used to pick only variables that were relevant to the model. A A total of 260 participants were randomly selected and included in the final analysis. The average age of study participants was 58.4 years (s.d. = 11.8), with the majority being females, 160/260 (61.5%). More than half of the participants, 159/260 (59.2%) in this study were earning the lowest of incomes (less than Botswana Pula [BWP] 3000 per month/approximately $277.05). The rest of the socio-demographic characteristics is shown in The majority of study participants were documented to have type 2 DM, 251/260 (96.5%). In contrast, over two-thirds of participants had been diabetic for 5 years or more, 175/260 (67.3%). Over half of the participants were either overweight or obese, 145/260 (55.8%) whereas 74.6%, 77.3% and 72.0% of study participants were HIV-negative, hypertensive and with at least one documented diabetic complication, respectively. Peripheral neuropathy and diabetic eye complications were the most commonly documented complications accounting for 60.8% and 57.7%, respectively .The mean glycaemic control (HbA1c) of the study participants was 7.4% (s.d. = 6.5% – 8.8%) with 162/260 (62.3%) of participants having suboptimal or poor glycaemic control .The prevalence of depression as measured by PHQ-9 was found to be 30.4% (79 participants). Beck’s Depression Inventory-II that was administered for the last 100 participants revealed a prevalence of depression of 27.0% . There wp-value = 0.002). Depression was more likely to be found among participants with lower education status, with the likelihood of being depressed decreasing in more literate participants. Participants with a tertiary education qualification were significantly associated with less depression . Participants who were never married were more likely to be depressed compared with those who were married (77% vs. 65.5%); however, the difference was not statistically significant . In contrast, analysis of the level of income showed participants who earned the lowest income (less than BWP3000 per month/approximately $277.05) were more likely to be depressed as compared with counterparts who earned more monthly income (BWP3000–BWP8000/approximately $277.05–$738.79 and more than BWP8000 (approximately $738.79); . The association between level of income and depression status was, however, not statistically significant . In contrast, participants who recorded high SBP of ≥ 140 mmHg were less likely to have depression as compared with those who recorded normal SBP . The rest of the studied clinical characteristics including duration of DM, a modality of treatment, number of documented diabetic complications, history of hypertension, HIV status and BMI were not associated with depression (table not shown).Participants with three or more diabetes-related hospitalisations were significantly more likely to be depressed compared with those with fewer diabetes-related hospitalisations . The rest of the diabetic complications was not associated with depression status (table not shown).Participants with no documented peripheral neuropathy were less likely to be depressed as compared with those with documented peripheral neuropathy . Glycaemic control using either fasting blood glucose or random blood glucose was not associated with depression status .There was a tendency of patients being more depressed as HbA1c increased. Patients with poor glycaemic control (HbA1c) >9% were statistically more likely to be depressed compared with those with good glycaemic control in bivariate analysis  and three or more diabetes-related hospitalisations  were significantly associated with having depression. Systolic blood pressure of ≥ 140 mmHg remained statistically significantly associated with less proportions of depression  between PHQ-9 and BDI II as revealed in the scatter plot in r) = 0.79 , indicating that the two tools have comparable convergence.The Cronbach’s alpha of 0.896 was obtained when comparing PHQ-9 and BDI II (Table not shown) indicating that the two tools have good internal consistency. There was a strong positive correlation ,36 Kenya (20.9%),34 Malawi (18%)33 and some Non-African countries: Jordan (19.7%)38 and Australia (20.8%).60 The wide variation in prevalence of depression between this study and others may be explained by several factors including differences in socio-demographic factors, study designs, depression assessment tools and sample size differences. Differences in culture and religion may also contribute to variations in prevalence of depression between countries.62 Societal norms such as focusing on physical symptoms of depression only with less emphasis on emotional aspects may also explain some differences.62 Evidence also shows that illness perceptions and thus their expressions may differ across cultures.62 In some societies and especially for older people, being depressed is considered as ‘normal’.62The prevalence of depression in our study in Botswana that used PHQ-9 cut-off ≥ 10 was found to be 30.4% indicating that almost a third of screened patients were depressed. This is almost similar to the findings of a recent meta-analysis of 248 studies showing a worldwide prevalence of 28%.14 Mental health problems including depression are more commonly observed in females than males in various communities and social contexts.25 This may be because of the burden brought by their multiple roles and responsibilities, lack of employment, sex discrimination and associated factors such as gender-based violence that contributes to women’s poor mental health.63Female gender was found to be significantly associated with depression both in bivariate and multivariate logistic regression. Similar findings have been observed across the majority of other studies.30 Education is thought to improve or expose an individual towards a better understanding of disease mechanisms and complications, leading to improved adherence towards disease treatment for better health outcomes. However, after a stepwise multivariate analysis, this effect was not observed; a possible explanation for this being that the sample size for the study might have been small to detect a difference in a multivariate model.Bivariate analysis revealed that higher education levels decreased the odds of being depressed; this is similar to several other studies.64 and South Australasia.25 However, it contrasts with a cross-sectional study among hospitalised patients with Type 2 DM at a tertiary hospital in Saudi Arabia, which found an increased risk of depression with older age.28 The difference may be because of different patient population characteristics.Age was not associated with depression in this study. This finding is similar to what was observed in other studies performed in Nigeria64 Single participants appeared to be more likely to be depressed as compared with those married in this study; however, the association was not statistically significant. The lack of significant association between marital status and depression found in this study is similar to findings from other studies.29Previous studies consistently showed that being single or unmarried is associated with an increased likelihood of having depression.29; however, this was not statistically significant probably because of sample size effect. It should also be observed that other studies that used smaller sample sizes than this study in Nigeria64 and Morocco65 also found no association between level of income and depression.A higher level of income in this study appeared to be associated with less likelihood of having depression; this is similar to previous studies66 This association may imply hospitalisations for acute complications such as diabetic ketoacidosis (DKA) or hyperosmolar hyperglycaemic state (HHS) precipitated by poor self-care in diabetic patients. Hospitalisation may also be related to chronic complications arising from poor glycaemic control observed in these depressed patients.Bivariate and multivariate analyses showed a significant association between being depressed and an increased number of DM-related hospitalisations. This was consistent with one large study in Australia.67 As the association of blood pressure and depression was only significant for SBP, but not DBP in our study, future studies must be conducted on this aspect to enhance our understanding.Bivariate and multivariate analysis revealed an inverse relationship between SBP control and depression. The results of this study are similar to what was reported in a study in Norway, which showed that depressed patients not on antidepressants were likely to have lower blood pressure (both DBP and SBP) than their controls.12 most of the studied DM complications in this study were not associated with depression. The most likely explanation for this is that the real extent of diabetic complications might have been underestimated because the study only counted documented complications; no active physical examinations, investigation or screening were performed on that respect. Other studied clinical characteristics included duration of diabetes, modality of diabetes treatment and history of hypertension and BMI. All these variables were not associated with depression contradicting previous studies that showed duration of diabetes, being on insulin and BMI to be associated with depression.23 In contrast, lack of association between duration of diabetes and insulin use in this study is consistent with a study carried out in Pakistan.24Although the presence of diabetic complications has been shown to be associated with depression68 it was important to assess the significance of HIV status as a contributor to depression. Human immunodeficiency virus infection was not associated with depression in this study. A possible explanation for this finding is that patients who attended at the tertiary diabetic clinic were well-controlled immunologically and virologically and hence less likely to be depressed compared with those 18–49 years recruited in a population-based study in districts for the previous study of depression among patients living with HIV in Botswana. Likewise, this study recruited fewer HIV-infected patients and hence may be less powered to detect any association. This lack of HIV association was also observed in the Kenyan study.34Given the high prevalence of HIV in Botswana and the fact that a previous study indicated over a quarter of studied participants living with HIV to be depressed,11 Glycosylated haemoglobin (HbA1c) of more than 9% was associated with depression; although this was not significant in multivariate analysis, it is of clinical significance and should be explored further in future local studies with larger sample sizes. Depressed mood can affect glycaemic control through at least two plausible mechanisms: either through changes in self-care or through counter-regulatory hormones or physiological disturbances. Several studies have shown an association between levels of depression and diabetes self-care. Ceichanowski et al.69 found that depression was associated with poorer diet, medication adherence and physical activity levels. Similarly, Lustman et al.8 found that depression was associated with poorer blood glucose self-monitoring. Depression-associated non-adherence was also observed in a meta-analysis of 47 studies.70 This, therefore, suggests that depressed mood could reduce appropriate self-care behaviours, which would negatively impact glycaemic control.Our bivariate analysis findings confirm and extend findings of previous studies by showing that depression is significantly associated with poor glycaemic control. This is consistent with numerous studies.Reliability analysis comparing PHQ-9 and BDI II revealed good internal consistency, and there was a strong linear positive correlation between the two depression screening tools. The PHQ-9 categorised a slightly greater proportion of patients with major depression than the BDI-II. These results indicate that despite both tools not being validated in this setting they can both be used to screen for depression among patients with DM in Botswana.The cross-sectional nature of the study does not provide a causal. Furthermore, the study may be prone to recall and reporting bias because some data were collected based on past and self-reported information. This could affect the nature of the relationships between depressive symptoms on the one hand and self-reported complications or medications on the other. Moreover, as the data were gathered from a single facility, findings may not be generalisable to all DM patients in Botswana, more especially those in the private sector or those in rural areas with no comprehensive diabetic care. In contrast, patients referred or being managed at this tertiary facility are those who cannot be managed at primary care clinics because of poorly controlled DM and that may have an impact on the experience of depression. This study excluded patients aged 18–20 years who also fall within the consenting age in Botswana; this is because most of the patients in this age group continue to follow up for their diabetic care at the adolescent clinic in the main referral hospital in Gaborone, Botswana. Despite the stated limitation, this is the first study to explore depression among DM patients in Botswana, and it offers important knowledge to build on future-related studies in Botswana and similar settings in SSA.The results of this study have shown that depression is a fairly common and under-recognised comorbidity among patients with DM in Botswana. It is an important piece of information to diabetes specialists, physicians, psychiatrists and general practitioners attending patients with DM to emphasise the need for routine screening of depression among patients with DM to enable early detection and treatment. Patients at risk for depression, including females, poor glycaemic control, those with three or more diabetes-related hospitalisations and lower SBP, should be prioritised. There is a need for future prospective and interventional studies across multiple facilities in Botswana to enhance more understanding of depression and its effect on morbidity and mortality of DM patients. Patient Health Questionnaire-9 depression screening tool was shown to have good internal consistency and strong positive linear correlation when compared with BDI II supporting its use in future studies.


This parasitism by PLE is such that it abolishes the production of ICP1 progeny and provides a defensive boon to the host cell population. In response to the severe parasitism imposed by PLE, ICP1 has acquired an adaptive CRISPR-Cas system that targets the PLE genome during infection. However, ICP1 isolates that naturally lack CRISPR-Cas are still able to overcome certain PLE variants, and the mechanism of this immunity against PLE has thus far remained unknown. Here, we show that ICP1 isolates that lack CRISPR-Cas encode an endonuclease in the same locus, and that the endonuclease provides ICP1 with immunity to a subset of PLEs. Further analysis shows that this endonuclease is of chimeric origin, incorporating a DNA-binding domain that is highly similar to some PLE replication origin-binding proteins. This similarity allows the endonuclease to bind and cleave PLE origins of replication. The endonuclease appears to exert considerable selective pressure on PLEs and may drive PLE replication module swapping and origin restructuring as mechanisms of escape. This work demonstrates that new genome defense systems can arise through domain shuffling and provides a greater understanding of the evolutionary forces driving genome modularity and temporal succession in mobile elements.Mobile genetic elements, elements that can move horizontally between genomes, have profound effects on their host's fitness. The  Mobile genetic elements (MGEs), genetic units capable of spreading within and between genomes, are key mediators of evolution. MGEs differ vastly in size and complexity. At one end of the spectrum are homing endonucleases (HEGs). These single-gene MGEs occur in diploid loci and cleave cognate alleles so that their coding sequences can serve as templates for recombinational repair . At anotRecent work has led to a deeper appreciation of the extensive entanglement between MGEs and genome defense functions. MGEs frequently encode defense modules to prevent infection by viruses or other nonvirus MGEs . Such moVibrio cholerae between the bacteriophage ICP1 and its own parasite, the phage-inducible chromosomal island-like element (PLE)  . Upon innt (PLE) , and is nt (PLE) . PLE excnt (PLE) . Similarnt (PLE) , PLE gennt (PLE)  stronglynt (PLE) . Thus, PTrue to the ‘guns for hire’ model, ICP1 has co-opted a genome defense system to protect itself from PLE. Many ICP1 isolates encode a CRISPR-Cas system that can destroy PLE within the infected cell and restore ICP1 reproduction . CRISPR-V. cholerae genomes. There is a pattern of temporal succession, where one PLE will dominate in sequenced genomes for a time before being supplanted by another PLE  determined host range in ICP1 isolates that lack CRISPR-Cas. It has long been recognized that phages are mosaic entities composed of functional gene neighborhoods, and syntenic neighborhoods of divergent sequence may fulfill analogous functions . Previougp88 for its location in ICP12001, the original sequenced ICP1 isolate  encoding gene. A gene encoding a Bro-N domain (pfam02498) and a KilAC domain (pfam03374) occurs adjacent to gp88. Their positions and putative annotations suggest that these divergently transcribed genes may have a regulatory function. In one sequenced ICP1 isolate with a functional CRISPR-Cas system  containing protein. This domain is a member of the GIY-YIG endonuclease domain superfamily, suggesting that Gp88 may be a nuclease. We aligned the Gp88 T5orf172 domain with GIY-YIG domains from endonucleases that had been biochemically characterized and structurally resolved  predicted to abolish Gp88’s nucleolytic activity  V. cholerae strain was susceptible to all ICP1 variants, and the ICP12006 ∆CRISPR variant was restricted by all PLEs  to which RepA binds covaried, with the PLE1, 4, and 5 sequences clustering together as one group, and the PLE2 and 3 sequences clustering as another . In these five strains, we discovered variation in a 67 bp stretch covering the iterons that results in several nucleotide changes. Within the first iteron, there is an A to T transversion, and starting at that transversion, the next 42 bp are duplicated and replace the sequence that is normally downstream  and tested its sensitivity to Odn in comparison to the WT PLE1 ori sequence. The PLE1Mut sequence was notably less susceptible to cleavage by Odn than the WT allele, but some cutting of the PLE1Mut probe at the highest concentration of Odn was apparent  cells, it was previously found that multiple spacers were needed to completely abolish PLE replication, and PLE transduction could still be detected from single-spacer ICP1 infections  ICP1 isolates are able to plaque on PLE2 when CRISPR is deleted . WhateveMut variant is able to escape Odn activity, given that the new iteron sequences are largely similar to those that exist in PLEs 1, 4, and 5. The most notable differences are an extra T/A base between the inverted sub-repeats in both the second and third iterons, and a reduction of space between the second and third iterons (now 5 bp instead of 14 or 16 bp) (It is somewhat surprising that the PLE1r 16 bp) . These cr 16 bp) . It is pr 16 bp) , making Alternatively, Odn cleavage might require recruitment of multiple Odn proteins, spaced a certain distance apart. RepA is thought to bind as dimer , but at V. cholerae-ICP1-PLE arms race. To defend itself against ICP1, V. cholerae makes use of the PLE, a selfish MGE. To protect against PLE, ICP1 has repurposed a nuclease domain from its own HEG parasites. While this recruitment of MGEs for antagonistic functions fits nicely within the ‘guns for hire’ model of MGE evolution, we see an interesting twist where it seems the horizontal gene transfer of DNA-binding domains is a central mediator of the PLE-ICP1 conflict. Recently, it was found that PLEs reduce ICP1 capsid gene expression through use of a regulator that resembles the DNA-binding domains of ICP1-encoded HEGs , 25 μg/ml chloramphenicol (Escherichia coli), and 100 μg/ml streptomycin. A detailed list of all strains used throughout this study can be found in the Key resources table.V. cholerae for 10 minutes at room temperature before the mixture was added to molten LB soft agar (0.5%) and poured onto 100 mm × 15 mm LB agar plates. Plaques were counted after overnight incubation at 37°C. Prior to phage infection for purposes of quantification or qPCR or spot assay analysis, V. cholerae was grown on plates overnight and then inoculated into 2 ml LB liquid cultures. Liquid cultures were grown to an OD > 1, then back diluted in fresh media to OD600 = 0.05, and then grown to OD600 = 0.3, at which point they were infected.Phage titers were determined using a soft agar overlay method wherein ICP1 was allowed to adsorb to V. cholerae mutants were generated through natural transformation as described previously  qPCR reactions. For assays involving induction of repA, 2 ml cultures were grown with 1.25 μg/ml chloramphenicol for plasmid maintenance and induced for 20 minutes prior to infection using a final concentration of 1.5 mM theophylline and 1 mM IPTG starting at OD600 = 0.17. All conditions were tested in biological triplicate, and each reported data point is the mean of two technical replicates. A single primer set (Key resources table) that amplifies a conserved region in all PLEs was used to detect PLE replication by qPCR.qPCR experiments were performed as previously described . BrieflyE. coli BL21 cells containing a His6-SUMO fusion to WT or E185A Gp88 were grown to OD600 = 0.5 at 37°C and induced with IPTG to a final concentration of 0.5 mM. The culture was grown for 2 hr and harvested by centrifugation at 4000×g for 20 minutes. The pellet was resuspended in lysis buffer  and sonicated. Cell debris was removed by centrifugation . The lysate was applied to a HisTrap HP column (Cytiva). The column was washed with wash buffer , and a high salt wash  was used to remove residual DNA. The protein was eluted using an elution buffer , and then the eluate was applied to a HiTrap Heparin HP column (Cytivia) for further purification. Following elution from the HiTrap Heparin column, the protein was dialyzed using a 10k Slide-A-Lyzer Dialysis cassette (Thermo Fisher) in 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM dithiothreitol (DTT). Concomitant with dialysis, the His6-SUMO tag was cleaved using SUMO protease. The SUMO tag was removed using Dynabeads (Invitrogen).All genomes were visualized and compared in CLC Main Workbench 7. Multiple sequence alignments were performed using the Multiple Sequence Alignment (MUSCLE) tool with default settings . The phy2, 50 mM NaCl, 1 mM DTT reaction buffer. Reactions proceeded at 30°C for 30 minutes, and were visualized on 0.8% agarose gels ran at 80 V for 30 minutes, and stained with GelRed (Biotium). For smaller probes ; Edze Westra (Reviewer #3).Thank you for submitting your article "A chimeric nuclease substitutes CRISPR-Cas: A phage weaponizes laterally acquired specificity to destroy subviral parasites" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Streamline the introduction to include only information critical to understanding how your work advances the field.2) An SDS-PAGE of the purified WT and mutant proteins is required to demonstrate purity and help support the conclusion that the Odn nuclease alone is responsible for cleavage of the PLE.3) Clarify inconsistencies and variabilities observed in the nuclease activity assays. For example, Figure 4d shows PLEs 1, 4, and 5 are cleaved to completion by WT Odn, while in Figure 6c and its supplements, the WT shows very little cleavage activity against PLE1. Different lengths of the substrates will shift the molar ratio of enzyme to substrate, and this impact the nuclease activity. Cleavage efficiencies must be quantified and normalized.4) Perform plaque assays for the ori mutant that renders this PLE resistant to Odn Figure 6c. This will be similar to the presentation of data in Figure 5a.5) Please specify that the evidence for Odn activity driving evolutionary change in the PLE is anecdotal.6) Sequence Odn cleavage products and map the precise location of these cuts.Reviewer #1:Barth et al. discover and characterize a new mechanism by which bacteriophage ICP1 defends itself against its own molecular parasites, the PLEs. PLEs are short stretches of DNA found in ~15% of sequenced epidemic strains of V. cholerae which hijack the structural components of bacteriophage ICP1 to facilitate their own spread-through this process, PLEs promote survival of the bacteria by preventing the spread of an ICP1 infection. Therefore, the interplay between ICP1 and its PLEs have significant impacts on Vibrio pathogenesis, highlighting the importance of this work. Previous research from this group has shown that some ICP1 variants harbor a CRISPR-Cas immune system, which these bacteriophages use to target and degrade PLEs. However, not all ICP1 variants possess CRISPR-Cas systems, and yet retain resistance to some PLE variants, suggesting the presence of other anti-PLE immune systems encoded in the bacteriophage genome. In this study, the investigators seek to understand how an ICP1 variant devoid of a CRISPR-Cas system can still defend against a subset of PLEs.StrengthsA rigorous set of genetics experiments were applied to reveal conserved functional aspects of PLE replication  as well as the genetic element in ICP1 required for anti-PLE activity, called gp88 . Based on sequence alignments and preliminary biochemical characterization, the gp88 gene product was shown to facilitate nuclease activity against PLE variants that are sensitive to ICP1 . Genetics was also successfully applied to narrow down the region within the PLE which is targeted by the gp88 (Odn) nuclease . Finally, a natural variant of PLE1 that seems resistant to Odn activity was discovered within a V. cholerae isolate from Pakistan , providing a beautiful example of how the ongoing conflict between host and parasite drives genetic diversification of both parties. Overall, the major conclusions in the paper are well-supported by the data, the manuscript is clearly written, and the study gives important insights into this system.WeaknessesThe biochemical characterization of Odn is preliminary and needs more rigorous experimentation.Comments for the authors:1. An image of the purified proteins, both WT and mutant variants, , should be displayed to demonstrate their purity and help support the conclusion that the Odn nuclease alone is responsible for cleavage of the PLE. The methods describe three separate chromatography steps, so it is assumed that the proteins are relatively pure, but a gel should be shown.2. The mutant version of Odn should be subject to the same level of scrutiny as the WT to solidify the conclusion that Odn is alone responsible for PLE cleavage, at least in the beginning. For example similar assays could be performed with the mutant as with WT in Figures 4c and Figure 5a.3. There seems to be some inconsistencies/variabilities in the biochemical assays. For example, Figure 4d shows PLEs 1, 4, and 5 are cleaved to completion by WT Odn, while in Figure 6c and its supplements, the WT shows very little cleavage activity against PLE1. Some quantification will help the reader to understand the extent of the variation in the nuclease activity. Along the same lines, different lengths of substrates were used in these assays, which will shift the molar ratio of enzyme:substrate and impact the nuclease activity. Some sort of normalization across the assays will allow the reader to compare the levels of nuclease activity from gel to gel and assess how each new variable affects the activity.4. To come full-circle, sequencing of the cut products would go a long way to support the genetics evidence for Odn targeting within the PLE origin and map the precise location of the cut(s).Reviewer #2:Vibrio cholerae. Past work from the Seed lab  had demonstrated that some ICP1 isolates  encoded their own type I-F CRISPR-Cas system to target PLEs. However, they also previously observed  that older ICP1 isolates that lacked CRISPR-Cas (2001-2011) could still plaque on some of the strains containing PLEs. This work sought to elucidate the mechanisms by which a CRISPR-less ICP1 isolate from 2001 could successfully overcome certain PLE variants, but not others.Barth et al. report the discovery of a novel endonuclease effector employed by certain lytic vibriophages (ICP1) to overcome viral satellites called PLEs present in ~15% of sequenced epidemic Through a set of simple yet elegant experiments that directly tested their hypotheses, the authors convincingly show that ICP12001 encodes an endonuclease  that allows it to successfully infect V. cholerae containing certain PLEs. Interestingly, Odn contains two domains-the C-terminus resembling a homing endonuclease (HEG), and the N-terminus resembling the DNA-binding domain of the replication initiator RepA of PLEs 1, 4, and 5. The authors speculate that Odn might have arisen from domain shuffling between ICP1-encoded HEGs and PLE-encoded RepA, although how exactly this occurred is unknown.The major strengths of this work are the novelty of the findings and its logical clarity. With a series of incisive experiments, the authors deduced how Odn works. By constructing ICP1 mutants that lacked Odn activity, the authors showed that Odn is necessary for plaque formation on V. cholerae containing PLEs 1, 4, or 5. Further, they show using qPCR that plaquing efficiency on PLE1 is correlated with Odn-mediated repression of PLE1 replication. In the future, it will be interesting to know if Odn alone is sufficient to overcome PLE, and whether the adjacent genes broN and gp90 play any regulatory or accessory role in Odn function.Next, the authors compared the genomes of all five PLE variants and found that the ori and RepA sequences cluster into two distinct groups-PLEs 1/4/5 and PLEs 2/3. This potentially explained why ICP12001 can plaque on strains containing PLEs 1, 4, and 5, but not PLEs 2 or 3, leading the authors to hypothesize that Odn recognizes and binds to the origin of replication of PLEs 1/4/5, but not PLEs 2/3. The Seed lab previously showed that non-replicating PLE mutants lacking ori could still restrict ICP1 . Exploiting this experimental tool, they showed here that ICP12001 could no longer plaque on strains containing PLE mutants lacking ori. Finally, by re-constituting the ori of PLE1 into PLE2, the strain is now sensitized to Odn activity upon ICP12001 infection. This decisive result strongly suggests that recognition of ori sequences is required for Odn-mediated plaquing.Finally, using an in vitro cleavage assay, the authors show that purified Odn cleaves PCR products amplified from the non-coding region containing the ori from PLEs 1/4/5, but not PLEs 2/3, consistent with the in silico prediction that the C-terminus of Odn resembles putative HEGs in ICP1. The authors further narrow down the region of specificity of Odn action to a set of iterons  and demonstrate using a PLE1 mutant lacking these sequences that iterons are necessary for Odn-mediated cleavage and plaquing by ICP12001. Surprisingly, there is only a 2-bp difference between PLEs 4 and 5, yet the cleavage pattern looks different between the two, raising the question of whether a more complicated mechanism of recognition and cleavage is in place.Finally, the authors address a crucial aspect of any host-parasite arms race-how can PLE escape from Odn-mediated restriction? They reason that simple deletions of the replication origin would not likely be selected for as a long-term solution, given its essentiality for PLE reproduction. Because of this, the authors speculate that Odn selected for domain shuffling events in PLE RepA, resulting in the two distinct clusters of PLEs 1/4/5 and PLEs 2/3. Although each PLE variant is remarkably conserved, the authors found an instance where PLE1 contained variation in its iteron sequences. They tested this variant and found that Odn was unable to cleave, suggesting that this variant PLE1 was likely resistant to infection by ICP12001, although they did not perform a plaque assay to confirm this.In conclusion, the authors complete a thorough characterization of Odn as an anti-PLE effector in ICP1. This work presents a key advance in our understanding of ICP1-PLE dynamics and brings broader insight to the roles of lateral gene transfer in the acquisition of novel weaponry in the host-parasite arms-race.Comments for the authors:Experimental:Figure 1: can Odn be re-constituted into a CRISPR-less ICP1 and restore plaquing against PLE1 to demonstrate sufficiency?Figure 6: In addition to the cleavage assay, to complement the in vitro data with in vivo results, the authors should perform plaque assays on PLE1mut for ICP12001 . It would be expected for ICP12001 to no longer be able to plaque given that Odn cannot cleave.Optional: it would be interesting to see if PLE escapers could arise naturally during an infection experiment and to determine what these mutations were. For example, you can infect a strain containing PLE1 with ICP12001 at very high MOI to lyse a whole plate. Are there any bacterial-insensitive mutants (BIMs) that form colonies? If so, are there any mutations in ori (and/or compensatory mutations in RepA), or perhaps even more strikingly, any restructuring of the iteron? This experimental evidence, together with the naturally occurring PLE1mut variant observed in Figure 6, would bolster the claim that Odn may be a driving force for the diversification of the PLE replication origin.Text edits:Title: the title, although descriptive, is a bit long and convoluted without having read the paper first. I would consider simplifying it to a single clause and remove jargon like "laterally acquired specificity"Introduction: the description of MGE and HGT in multicellular organisms in the first paragraph is not necessary to understand this work and possibly too much information for the reader.Figure 2: although this is beyond the focus of this work and does not need to be addressed in the text, it does not escape my attention that PLE3 is able to evade both CRISPR (no targeting spacer) and Odn (ori not recognized). Is there a contemporaneous ICP1 isolate that is able to plaque on PLE3, and if so, is there a different effector in the same genomic region between gp87 and gp91?Figure 3: is there a working model for how PLEs recombine (if they even do) to form new variants? Are their isolates of V. cholerae that contain multiple PLEs?Lines 342-343: "Remarkably, Gp88's own N-terminal domain is 61% similar to 93% of PLE1's RepA_N domain".This sentence is a bit misleading, given that it's only 42% identical (which is still quite similar). The inclusion of "similar residues" in the comparison indeed yields 61%, but this factor in the calculation should be explicitly stated in the text.Figure 4D: There appears to be two cuts for PLEs 1 and 5, but not 4? This is interesting, especially since there's only a 2-bp difference in the iteron sequences between PLEs 4 and 5 -is there any explanation for the differences between the cleavage pattern of the three PLEs at this time, and whether these provide any clues as to Odn's mechanism of action?Discussion:Lines 551-552: what are some examples of auxillary genes that are present between CRISPR-less ICP1 vs. CRISPR(+) ICP1? Are CRISPR-less ICP1 isolates on average just slightly smaller?Reviewer #3:Phage ICP1 infection can be blocked by PLE elements in Vibrio genomes, which use phage capsids for their own dissemination, but ICP1 has in turn evolved mechanisms to overcome PLE-mediated defence/parasitism. Seed and colleagues have discovered and extensively characterised how a CRISPR-Cas system encoded by the phage can cleave the PLE element, leading to productive infections.Here, the authors aimed to explain why an ICP1 genotype that naturally lacks CRISPR-Cas, and that used to be very common before CRISPR-carrying variants appeared, can infect Vibrio cells that certain PLE variants. Specifically, their previous work found that this ICP1 variant can infect cells that carry the two oldest PLE variants, PLE5 and PLE4, as well as the most recent variant PLE1 .They found that this older ICP1 genotype carries an alternative nuclease  that cleaves the origin of replication of some PLE elements. This nuclease gene is located in the same locus as the CRISPR-Cas genes in more recent ICP1 genotypes. This newly discovered nuclease carries an N-terminal DNA binding domain that has high sequence similarity to RepA, which binds the origin of replication, and a C-terminal nuclease domain that is similar to a ICP1-encoded family of putative homing endonucleases (HEGs), and that is responsible for cleavage of the origin of replication. Different PLE variants (PLE1-5) vary with respect to their RepA and origin of replication sequences – specifically, the authors show that PLE1, 4 and 5 share RepN and Ori sequences, explaining why the ICP1 variant that carries Odn can infect cells that carry these PLE elements, but not cells that carry PLE2 or PLE3.The conclusions of this paper are well supported by comparative genomics, elegant reverse genetics analyses and biochemical assays. The authors generate all the relevant KO variants of ICP1 and PLE to demonstrate the genetic interaction between Odn of ICP1 and the Ori of PLE, including ori-swapped mutants of PLE, where PLE2 carries the ori of PLE1, leading to a lack of protection of the host from phage infection. Finally, the authors purified Odn and demonstrate that it can cleave the Ori sequences in vitro, but not random sequence, and a catalytic mutant is unable to cleave either (and the same mutation leads to PLE sensitivity in vivo). This binding and cleavage is specifically directed towards a set of semi-palindromic sequences, known as iterons.All in all, this is a very thorough and complete study that provides novel and deep insight into the ongoing coevolutionary interaction between PLE and ICP1.Comments for the authors:This is a very nice study – I enjoyed reading the paper. I only have fairly minor comments/suggestions:1. This is a matter of preference in style, but in my opinion the introduction and discussion could be much tighter. The first 3 paragraphs of the introduction provide a lot of background that is not essential for this study, perhaps more appropriate for a broad review, and could be trimmed substantially. The discussion is2. I'm left wondering whether BroN and gp90 are essential for the activity of Odn. Have you made knockouts of these genes ? Or alternatively, have you expressed Odn from a plasmid in cells with PLE and looked for evidence of cleavage  ? You mention they likely have regulatory roles – what is the evidence in support of this idea ?3. Figure 3A shown the ori and RepA-N sequence variation between PLE1,4,5 on the one hand, and PLE2,3 on the other. Supplement 1 only shows the alignment of the conserved c-terminus. Can you please add the N-terminal sequence to enable more detailed examination of the level of sequence variation / conservation between the different groups?4. the similarity of the nuclease domain to the putative homing endonucleases in ICP1 is intriguing, and provides a plausible explanation for the origin of the nuclease domain. How common are these homing endonucleases? Can the authors make a tree of these nuclease domains to see if this Odn nuclease domain fall inside the tree? Or do you not have enough sequences for this / is sequence divergence too high?5. Figure 4 D – nice to see the cleavage activity of gp88/Odn. It should not be too hard to identify the cleavage site using Sanger sequencing . Have you tried this?6. Line 456-459 – I don't think the data are sufficient to draw this conclusion; even if the mutation provides resistance to Odn, it is impossible to work out from these data whether or not the Ori mutations are adaptive, and if so, whether or not this is because of ICP1 infections. This statement therefore needs to be further toned down in my opinion.7. Line 544 – the Gomez et al. Science paper from the same group would also be appropriate to cite. Essential revisions:1) Streamline the introduction to include only information critical to understanding how your work advances the field.We have streamlined the introduction removing several examples.2) An SDS-PAGE of the purified WT and mutant proteins is required to demonstrate purity and help support the conclusion that the Odn nuclease alone is responsible for cleavage of the PLE.We have added images of the SDS-PAGE gels of protein preparations used in in vitro assays as Figure 4—figure supplement 2.3) Clarify inconsistencies and variabilities observed in the nuclease activity assays. For example, Figure 4d shows PLEs 1, 4, and 5 are cleaved to completion by WT Odn, while in Figure 6c and its supplements, the WT shows very little cleavage activity against PLE1. Different lengths of the substrates will shift the molar ratio of enzyme to substrate, and this impact the nuclease activity. Cleavage efficiencies must be quantified and normalized.Mut at high concentrations of Odn, however, together with additional in vivo evidence (discussed in response to point 4), our data indicate that the mutant ori is indeed resistant to Odn-mediated cleavage.For Figure 6C we originally used a shorter substrate for both the WT PLE1 origin andthe mutant PLE1 origin because we had difficulty cloning the mutant origin sequence into the larger PLE1 context (since the mutant was identified in sequenced strains from other groups and not in our collection). We suspect you are correct that the loss of cutting was due to a decreased molar ratio of enzyme to substrate. We have now successfully generated the longer mutant PLE1 origin probe and have repeated the experiment using probes of the same length as in Figure 4D. These new data are shown in revised Figure 6C . With the longer probe we do see some cleavage of the PLE1We also want to point out that we do not consistently see complete loss of the uncleaved substrate of even the same probe, for example, compare the three replicates of Figure 4C  – we only saw complete loss of the uncleaved substrate for the PLE1 probe in one replicate. We have therefore replaced the gel in the main figure with one of the (former) supplementary gels to reflect that complete cleavage is not the outcome the majority of the time. We are unfortunately not able to explain this minor inconsistency, and given that we make no claims regarding cleavage efficiency in vitro (and do not feel comfortable doing so), we did not quantify cleavage efficiency. We hope that the combination of in vivo and in vitro evidence, which together tell the same story regarding cleavage specificity, is satisfactory.4) Perform plaque assays for the ori mutant that renders this PLE resistant to Odn Figure 6c. This will be similar to the presentation of data in Figure 5a.Mut is resistant to Odn(+) ICP1 as anticipated. To take this one step further, we also performed qPCR of the PLE1Mut strain during ICP1 infection and show that PLE1Mut replicates during infection and the copy increase is not impacted by Odn . Collectively, we feel these new data provide more support for our conclusion that this mutation is adaptive in the face of Odn.We have now completed these experiments, and the results are presented in Figure 6E. These data show that PLE15) Please specify that the evidence for Odn activity driving evolutionary change in the PLE is anecdotal.We have addressed this point: we have removed our original assertion that “These datasuggest that Odn imposes selective pressure on the origin of replication…” and substituted more cautious language, particularly with regard to the data in revised Figure 6: “These data demonstrate that PLE can escape Odn activity through subtle restructuring of the iterons, in addition to more extensive replication module exchange , and suggest that ICP1 defenses like Odn may select for diversification of the PLE replication machinery.”As well as in the discussion: “PLEs are able to escape Odn antagonism through mutation of their origin of replication and there is compelling evidence suggesting that PLEs have exchanged replicon modules for an alternative replication origin and initiation factor origin binding domain on at least two separate occasions.”We feel that we were also appropriately cautious in the language we used in parts of theoriginal submission (these examples remain unchanged in the revised manuscript):“The endonuclease appears to exert considerable selective pressure on PLEsand may drive PLE replication module swapping”;“It is possible that Odn selected for alternative replicon modules, leading to thedecline of PLE4 and succession of PLE2 that occurred in the early 2000s . […] This seems like a likely possibility for PLEs and ICP1 whose specific adaptations.”6) Sequence Odn cleavage products and map the precise location of these cuts.This was something we tried  prior to the initial submission, and in response to reviewer comments made several more attempts. Using Sanger sequencing of Odn cleavage products we were unable to make a clear determination of where cutting was taking place. In most cases we saw uncleaved substrate in our sequencing results (see response to point 3), even when no uncleaved product was visible on the gel and after gel extracting smaller cleaved products. Using higher protein concentrations and longer incubation times did not resolve this issue.In some of our attempts to address this point, chromatograms obtained by Sanger sequencing did suggest that Odn cleavage of PLE1 is occurring proximal to the poly-T centers of the first and second iterons, but these sequencing results were somewhat inconsistent, and we do not have a robust method of calling sub-sequence ends from the amplitudes of base signals. We therefore do not feel confident enough to include these results in the manuscript.We also attempted subcloning of cleavage products via TA cloning and blunt end cloning, but were unable to recover cleaved products through these methods.We have, however, taken additional steps requested by reviewer 1 – including cleavage assays with Odn* and other substrates (see below). Ultimately, although we do not know the precise location where cleavage is taking place, we feel that our in vivo and in vitro results are sufficient to demonstrate Odn targeting through the iterons. We agree that our biochemical characterization of Odn is preliminary and indeed include that this is an area that needs further study: “however the molecular details of Odn binding and catalysis remain to be elucidated.”Reviewer #1:[…] Comments for the authors:1. An image of the purified proteins, both WT and mutant variants, , should be displayed to demonstrate their purity and help support the conclusion that the Odn nuclease alone is responsible for cleavage of the PLE. The methods describe three separate chromatography steps, so it is assumed that the proteins are relatively pure, but a gel should be shown.We have added images of the purified protein as Figure 4 —figure supplement 2.2. The mutant version of Odn should be subject to the same level of scrutiny as the WT to solidify the conclusion that Odn is alone responsible for PLE cleavage, at least in the beginning. For example similar assays could be performed with the mutant as with WT in Figures 4c and Figure 5a.In figure 5A, we include Odn* at our maximum concentration of 500nM and detect no cleavage of the PLE1 probe. In response to this comment we have now tested Odn* against other probes (PLE4 and PLE 5) which were cut by Odn in Figure 4D. These new data are presented in Figure 4E  and show that 500nM Odn* does not cleave probes cut by Odn, consistent with the in vivo data and our conclusions.3. There seems to be some inconsistencies/variabilities in the biochemical assays. For example, Figure 4d shows PLEs 1, 4, and 5 are cleaved to completion by WT Odn, while in Figure 6c and its supplements, the WT shows very little cleavage activity against PLE1. Some quantification will help the reader to understand the extent of the variation in the nuclease activity. Along the same lines, different lengths of substrates were used in these assays, which will shift the molar ratio of enzyme:substrate and impact the nuclease activity. Some sort of normalization across the assays will allow the reader to compare the levels of nuclease activity from gel to gel and assess how each new variable affects the activity.Thank you for this suggestion. Please see our response to essential revisions point 3.4. To come full-circle, sequencing of the cut products would go a long way to support the genetics evidence for Odn targeting within the PLE origin and map the precise location of the cut(s).Please see our response to essential revisions point 6.Reviewer #2:[…] Comments for the authors:Experimental:Figure 1: can Odn be re-constituted into a CRISPR-less ICP1 and restore plaquing against PLE1 to demonstrate sufficiency?V. cholerae (which requires cloning and subsequent maintenance in E. coli and V. cholerae to generate the genome editing strain) so we have not been able to reconstitute its presence within the ICP1 genome.Thank you for the suggestion, reconstitution of Odn restoring ICP1 ∆CRISPR plaquing on PLE1 would strongly support our model and was in fact something we tried quite early on. Unfortunately we found that Odn in its native sequence context is too toxic to provide as an ICP1 editing template in Figure 6: In addition to the cleavage assay, to complement the in vitro data with in vivo results, the authors should perform plaque assays on PLE1mut for ICP12001 . It would be expected for ICP12001 to no longer be able to plaque given that Odn cannot cleave.Thank you for this suggestion. We have now performed these experiments and the results are presented in Figure 6E.Optional: it would be interesting to see if PLE escapers could arise naturally during an infection experiment and to determine what these mutations were. For example, you can infect a strain containing PLE1 with ICP12001 at very high MOI to lyse a whole plate. Are there any bacterial-insensitive mutants (BIMs) that form colonies? If so, are there any mutations in ori (and/or compensatory mutations in RepA), or perhaps even more strikingly, any restructuring of the iteron? This experimental evidence, together with the naturally occurring PLE1mut variant observed in Figure 6, would bolster the claim that Odn may be a driving force for the diversification of the PLE replication origin.V. cholerae with ICP1 are overwhelmingly receptor loss mutants . Further, while a selection method for PLE escape would be highly useful, the outcome of either ICP1 ‘winning’ or PLE ‘winning’ is cell death, which makes a selection of this sorts extremely challenging: PLE induction ultimately culminates in cell lysis, so even if PLE escape mutants were present, the bacteria harboring those mutant PLEs cannot be recovered. It is possible that PLE escapes (in the form of transductants) could be isolated, though we have previously shown that this does not happen with CRISPR(+) ICP1 , so we did not attempt it for Odn.This would indeed be very interesting. We did not attempt this; in our experience so far BIMs arising following infection of Text edits:Title: the title, although descriptive, is a bit long and convoluted without having read the paper first. I would consider simplifying it to a single clause and remove jargon like "laterally acquired specificity"Thank you for the suggestion. We have changed the title to: “A chimeric nuclease substitutes a phage CRISPR-Cas system to provide sequence specific immunity against subviral parasites.”We think this new title retains the information we wanted to convey while being more accessible.Introduction: the description of MGE and HGT in multicellular organisms in the first paragraph is not necessary to understand this work and possibly too much information for the reader.These examples, as well as few prokaryotic examples, have been removed to streamline the introduction.Figure 2: although this is beyond the focus of this work and does not need to be addressed in the text, it does not escape my attention that PLE3 is able to evade both CRISPR (no targeting spacer) and Odn (ori not recognized). Is there a contemporaneous ICP1 isolate that is able to plaque on PLE3, and if so, is there a different effector in the same genomic region between gp87 and gp91?V. cholerae strains.Previously we’ve shown that spacers against PLE 3 are functional when acquired by this phage isolate  The original isolate just happens to not have a spacer directed against PLE3. As for why this is the case, a possible explanation is that PLE3 appears to be much rarer than other PLEs, occurring in a relatively small number of sequenced gp87 and gp91 in sequenced ICP1 isolates – this analysis includes an expanded set of ICP1 isolates collected between 1992-2019 and is part of a review article in press . Of course we would be very intrigued to find isolates with different genes in that region of the ICP1 genome – we will see what we find in future sampling efforts.We have not yet seen anything besides gp88 or CRISPR-Cas in the region between Figure 3: is there a working model for how PLEs recombine (if they even do) to form new variants? Are their isolates of V. cholerae that contain multiple PLEs?The mosaic sequences of PLEs suggest that PLEs have recombined at some point, but we do not have direct evidence for this recombination taking place. To date, no sequenced isolates have been found to harbor multiple PLEs. Potential explanations for this could be that carriage of multiple PLEs is unstable, or that PLEs recombine following transduction of a PLE into a strain that already has a PLE.Lines 342-343: "Remarkably, Gp88's own N-terminal domain is 61% similar to 93% of PLE1's RepA_N domain".This sentence is a bit misleading, given that it's only 42% identical (which is still quite similar). The inclusion of "similar residues" in the comparison indeed yields 61%, but this factor in the calculation should be explicitly stated in the text.We’ve amended this line to say "Remarkably, Gp88's own N-terminal domain is 42% identical and 61% sequence similar to 93% of PLE1's RepA_N domain". (P5 Line 233)Figure 4D: There appears to be two cuts for PLEs 1 and 5, but not 4? This is interesting, especially since there's only a 2-bp difference in the iteron sequences between PLEs 4 and 5 -is there any explanation for the differences between the cleavage pattern of the three PLEs at this time, and whether these provide any clues as to Odn's mechanism of action?We apologize for any confusion regarding cleavage patterns. While it is true that there is only a 2-bp difference in the iteron sequence between PLEs 4 and 5, the probes used for our cleavage assays encompass a larger sequence harboring the origin of replication and contain numerous differences between the PLEs . Much of these differences consist of gain or loss of novel sequence. We designed our probes without considerable attention to where the iterons fell within the probes, and the PLE4 probe happened to have the iterons directly in its center. This is predicted to produce two cleavage products of the same size, which would appear as a single band on a gel .We realize that this is likely to be a point of confusion for readers so we now address this in the text (P5 lines 265-270) and have added a supplementary figure , showing that when different primers are used to amplify the PLE4 probe (resulting in the iteron sequence offset from the center of the probe) the cleavage products clearly run as two bands, as would be predicted.Discussion:Lines 551-552: what are some examples of auxillary genes that are present between CRISPR-less ICP1 vs. CRISPR(+) ICP1? Are CRISPR-less ICP1 isolates on average just slightly smaller?We discuss auxiliary gene diversity and total genome length across ICP1 isolates in a forthcoming review article that’s been accepted for publication .In summary, ICP1 isolates range in genome length from ~121kb to ~131kb, and the CRISPR-Cas system is ~7kb. Generally speaking yes, the isolates with the larger genomes tend to be CRISPR(+), but surprisingly, some of the largest ICP1 genomes lack CRISPR-Cas.Reviewer #3:[…] Comments for the authors:This is a very nice study – I enjoyed reading the paper. I only have fairly minor comments/suggestions:1. This is a matter of preference in style, but in my opinion the introduction and discussion could be much tighter. The first 3 paragraphs of the introduction provide a lot of background that is not essential for this study, perhaps more appropriate for a broad review, and could be trimmed substantially.We have streamlined the introduction.2. I'm left wondering whether BroN and gp90 are essential for the activity of Odn. Have you made knockouts of these genes? Or alternatively, have you expressed Odn from a plasmid in cells with PLE and looked for evidence of cleavage ? You mention they likely have regulatory roles – what is the evidence in support of this idea?We have noted that WT Odn is cytotoxic in PLE1(+) hosts, but felt that the in vitro cleavage assays and infection host range phenotypes were more compelling than this toxicity phenotype and did not pursue it further.broN-related gene and gp90 could yield interesting results, we do not think there’s enough space in this manuscript to also characterize their function.While investigating the roles of the gp88. Their positions and putative annotations suggest that these divergently transcribed genes may have a regulatory function.” We believe our language is more cautious than saying that they are likely regulators, and merely puts forward a model consistent with their annotations, location, and orientation. It is common for regulators to be divergently transcribed from the genes they control. The BroN domain is thought to be a DNA binding domain, and the KilAC domain is associated with transcriptional antitermination activity.In regard to these genes, we write “A gene encoding a Bro-N domain (pfam02498) and a KilAC domain (pfam03374) occurs adjacent to 3. Figure 3A shown the ori and RepA-N sequence variation between PLE1,4,5 on the one hand, and PLE2,3 on the other. Supplement 1 only shows the alignment of the conserved c-terminus. Can you please add the N-terminal sequence to enable more detailed examination of the level of sequence variation / conservation between the different groups?We have expanded the supplementary figure to incorporate the full coding sequence.4. The similarity of the nuclease domain to the putative homing endonucleases in ICP1 is intriguing, and provides a plausible explanation for the origin of the nuclease domain. How common are these homing endonucleases? Can the authors make a tree of these nuclease domains to see if this Odn nuclease domain fall inside the tree? Or do you not have enough sequences for this / is sequence divergence too high?2001 show noticeable similarity to Odn’s nuclease domain. This family of HEGs is discussed in a recent publication about PLE and ICP1 , a more detailed discussion of ICP1 HEGs and their potential role in ICP1 genome evolution will appear in a forthcoming review , and we’re continuing work to understand the relationship between different HEGs in ICP1 and related phages.Homing endonuclease are fairly common within lytic bacteriophages though the extent of this varies between phage clades. This family of T5orf172 homing endonucleases appear to be fairly common in ICP1 related phages, though only four HEGs in ICP1As requested, we’ve added a tree of the aligned HEG domains . It’s a small number of sequences that are short in length (~200aa), but we think the case for horizontal transfer of the Odn endonuclease domain is compelling.5. Figure 4 D – nice to see the cleavage activity of gp88/Odn. It should not be too hard to identify the cleavage site using Sanger sequencing . Have you tried this?We have! Please see our response to essential revisions point 6.6. Line 456-459 – I don't think the data are sufficient to draw this conclusion; even if the mutation provides resistance to Odn, it is impossible to work out from these data whether or not the Ori mutations are adaptive, and if so, whether or not this is because of ICP1 infections. This statement therefore needs to be further toned down in my opinion.Thank you for this suggestion. After reviewing what we had written, we agree that the original statement was over reaching. We’ve amended this passage to read:“These data demonstrate that PLE can escape Odn activity through subtle restructuring of the iterons, in addition to more extensive replication module exchange , and suggest that ICP1 defenses like Odn may select for diversification of the PLE replication machinery.”7. Line 544 – the Gomez et al. Science paper from the same group would also be appropriate to cite.Thank you for this suggestion, we have added this citation.


Horses are prone to develop a wound healing disorder on their limbs called exuberant granulation tissue (EGT). The exact mechanism for the formation of this tissue remains unknown but the inflammatory response is supposed to be an important contributing factor. In this article, we investigated this inflammatory response in both EGT wounds as well as in control horse wounds. In biopsies, we detected two types of immune cells: (1) immune cells involved in early inflammation (MAC387+ cells) and (2) immune cells important in the later phases of inflammation (CD163+ cells). We detected a higher number of immune cells in EGT wounds compared with the control wounds of 19 days old. This suggests that EGT wounds may not be able to proceed through further phases in the wound healing process or that the inflammation phase is prolonged. Exuberant granulation tissue (EGT) is a frequently encountered complication during second intention healing in equine distal limb wounds. Although it is still unknown what exactly triggers the formation of this tissue, previous research has revealed a persistent inflammatory response in these wounds. In this preliminary study we examined this inflammatory response in EGT-developing wounds as well as in experimental induced wounds. Immunohistological stainings were performed to detect primary inflammatory immune cells (MAC387 staining) as well as pro-resolution immune cells (CD163 staining). Our results show a significantly higher amount of MAC387+ and CD163+ cells in the fibrotic regions of EGT compared with the 19-day-old experimental wounds. This persistent high amount of fibrosis-promoting CD163+ cells in EGT suggests that the wound healing processes in EGT-developing wounds are arrested at the level of the proliferation phase. Wound healing is an essential process for the health of animals but can also be associated with severe complications. Veterinarians are frequently confronted with horses that have serious wound healing issues at the level of the distal limbs. Wounds at these locations are often accompanied by massive tissue loss and typically need to heal by second intention, i.e., through the initial formation of granulation tissue that fills the wound bed and the subsequent re-epithelialization and wound contraction ,2. GranuThe exact physiopathology of EGT remains unclear but comparative studies have already revealed that differences in the immune response have a big impact on the formation of EGT ,5. In coIn physiological healing wounds, the inflammatory response starts when immune cells such as neutrophils and monocytes are recruited to the wound bed. Monocytes can differentiate into macrophages. These highly plastic cells can change their phenotype and function throughout the wound healing process according to the stimuli present in the micro-environment . In the In this article, different immunohistological and histological stainings were performed to gain more insights in the inflammatory response occurring in EGT.Two types of wound healing samples were used, i.e., samples of physiological second intention healing experimental equine wounds as the control and exuberant granulation tissue from equine patients. The samples of physiological healing wounds were derived from a previous study (a non-contaminated control treatment) . The oriExuberant granulation tissue was collected from patients presented at the Faculty of Veterinary Medicine of Ghent University. To be included in this study, it was important that EGT material could be obtained as fresh as possible. Apart for one exception, the wounds were at least two weeks old. EGT included in this study was obtained from twelve different warmblood horses . The weight was recorded for most horses and varied from 340 to 635 kg. Circular wounds of 3.5 cm in diameter were created on the dorsomedial aspect of both metacarpi during an aseptic surgery. Both the skin and subcutis were removed using a scalpel. In the center of each wound, the periosteum was also removed over a circular area of 2 cm diameter with the aid of a sterile template. The underlying bone was curetted 15 times each in both a dorsopalmar and proximodistal direction. The wounds were first covered with a sterile non-adherent absorbent dressing , which was fixed using an elastic retention dressing . The next layer consisted of a standard cotton wool limb bandage and the top layer was an elastic bandage . All the samples used in the current study originated from the non-contaminated control group. The day after surgery the bandages were removed and one limb was assigned to the treatment procedure and the other to the reference treatment. All the samples used in the current study originated from wounds cured with the reference treatment. For this option, the wounds were covered with calcium alginate dressings  soaked with a 20 mL sterile saline solution (0.9% NaCl). This primary dressing was molded to the wound to minimize overlap with the surrounding skin and covered with an elastic retention dressing . A standard limb bandage was then applied, as described earlier. The primary wound dressing was calcium alginate for 9 days with bandage changes every 3 days. Afterwards, a hydrophilic polyurethane foam dressing  was applied, which was changed every 5 to 7 days. Eight millimeter punch biopsies (6 mm granulation tissue + 2 mm surrounding skin) were taken 6 and 19 days after the wound creation along the wound edge of the distal limb wounds and fixated in 10% neutral buffered formalin. These two timepoints were chosen because at these moments the acute inflammatory phase and proliferation phase, respectively, are at their maximum in full-thickness wounds on the limbs of horses ,19.EGT was collected from equine patients treated at the horse clinic for EGT formation in distal limb wounds . EGT wasAfter a fixation of approximately 24 h, the tissues were paraffin embedded using a tissue processor  and embedding station . Serial sections of 5 µm were cut using a microtome  and stretched in a warm water bath (48 °C). Subsequently, the sections were captured on adhesive slides  and dried overnight in an incubator at 37 °C.Two histological stainings were performed, i.e., the routinely used hematoxylin-eosin staining and the Masson trichrome staining known for its capability to detect collagen fibers. The hematoxylin-eosin staining was performed automatically by the Gemini AS automated slide stainer . For the Masson trichrome staining  the follOn two serial sections an immunohistochemical staining was performed using two different antibodies, i.e., CD163  and MAC387 . Clone AM-3K has been shown to recognize CD163, a commonly used marker for anti-inflammatory macrophages or pro-resolution macrophages . The MAC2O2 in methanol  to block endogenous peroxidase activity and incubated for 30 min with a 1/200 biotinylated rabbit anti-mouse secondary antibody . After washing, an additional incubation with Streptavidin-HRP (1/1000) was performed for 30 min . For visualization the slides were incubated with a DAB+ substrate chromogen system  for 5 min and subsequently counterstained with hematoxylin for 30 s. After rinsing the slides in tap water for 10 min the slides were dehydrated to xylene and mounted with DPX .The slides were deparaffinated using xylene and hydrated in a decreasing gradient of alcohol. After hydration in distilled water, antigen retrieval was performed using antibody-specific procedures. For the CD163 antibody, the slides were subjected to a heat induced antigen retrieval of 15 min at 110 °C in a decloaking chamber  using a target retrieval solution with a pH of 6 . For the MAC387 antibody an antigen retrieval with Proteinase K  for 15 min was performed. After the antigen retrieval, the slides were washed with a wash buffer  for 3 min before a hydrophobic circle was applied around the sections with a PAP pen . The sections were then blocked with 30% rabbit serum. Thereafter, the sections were incubated for 1 h with primary antibodies in a humified chamber at room temperature. The antibodies were diluted in an antibody diluent  at the following concentrations: CD163 1/1600 and MAC387 1/2000. After a wash step, the slides were incubated for 5 min in 3% HAll slides were photographed and scanned with a 20× objective using an Olympus BX 61 microscope and CellSens software . Subsequently, the pictures were opened and analyzed with QuPath software . First, With the aid of a multiple view window and morphological landmarks, the same regions were analyzed in the serial sections. In the case of an artifact or a tear in the tissue, these were omitted from the selection. That is why for every square not only the number of positive pixels was counted but also the exact surface of the selected area. When analyzing the results, the positive pixels counts were divided through this number and subsequently multiplied by 250,000 (=500 × 500) to take the differences in the analyzed surface area into account. For each tissue block the geometric mean was calculated from the two fibrotic regions and the two inflammation regions. Subsequently, these means were log transformed. https://CRAN.R-project.org/package=ggpubr) and ggplot2  packages, which allowed data visualization [p-value of <0.05 was considered significant.A statistical analysis was performed in RStudio (version 3.6.2) with the aid of the ggpubr  was observed between EGT-developing wounds and physiological healing wounds 19 days post-injury in both the inflammatory and fibrotic regions of the wound (p = 0.0043) but less in the inflammatory regions (p = 0.052)  B,C.p = 0.0079) between day 6 and day 19 post-injury in the fibrotic/deeper regions of the wound (p = 0.0094) in CD163 staining was seen between EGT wounds and experimental wounds 19 days post-injury (p = 0.13). CD163+ cells were more uniformly distributed in both the experimental wounds as EGT compared with the MAC387+ cells . No signhe wound . In the he wound . In the t-injury A,C. HoweOne of the main characteristics of EGT is the persistent inflammatory response in these wounds. This chronic inflammation may be one of the driving factors stimulating the formation and maintenance of EGT . In physThe inflammatory regions located close to the wound surface contained a lot of MAC387+ cells. The MAC387 antibody is a calprotectin-specific antibody that is expressed on granulocytes such as neutrophils, infiltrating monocytes and potential M1-like macrophages . This MAIn the inflammatory regions, the number of MAC387+ cells seemed to decline between day 6 and 19 after injury in physiological healing experimental wounds. However, in EGT the overall amount of MAC387+ cells remained high, indicative of a persistent inflammatory response in EGT. As the wound bed was still open in both the experimental and EGT wounds, it is not surprising that primary inflammatory cells remained present in the upper parts of the wound. In the fibrotic regions of the wound, the MAC387 staining was significant higher in EGT-developing wounds compared with the physiological healing wounds. This in indicative of a persistent inflammatory response in EGT-developing wounds.The CD163 staining in the inflammatory regions showed a high variability. This can probably be explained by the fact that this region was located near the wound surface and is strongly influenced by external factors including the degree of contamination and the amount of wound exudate.A difference was also observed in CD163 staining between the wound types in the fibrotic regions of the wound. Between day 6 and day 19 after injury, a significant drop in CD163+ cells was observed in the fibrotic regions of physiological healing wounds. CD163+ staining in EGT was more variable. Higher levels of CD163 cells were found in EGT wounds compared with the physiological healing wounds 19 days after injury but when the analysis was only performed on the six horses with ages in the same interval as the horses from the experimental study, the differences became less pronounced. This may indicate an age effect but the number of horses in each age interval was too small to verify this.In physiological wound healing, the number of M2-like macrophages declines when the wound transits from the proliferation to the remodeling phase. As the number of M2-like macrophages in EGT resembles more closely the physiological wounds of 6 days compared with the physiological wounds of 19 days, this may suggest that EGT consists of young fibrotic tissue stuck in the proliferation phase, which is unable to transit to the remodeling phase. This may also explain the excessive fibroplasia typically seen in EGT. Excessive M2 macrophage activation has been associated with scarring and fibrosis ,26. ThisHuman diabetic ulcers are characterized by a chronic but low grade inflammation similar to EGT wounds ,29,30,31It is of note to mention that the distinction between M1 and M2 macrophages is not strict and that these two types of macrophages need to be considered as the two extremes of a whole spectrum . For exaOne of the limitations of this study is the limited number of horses included in the study. Another limitation was the fact that EGT used in this preliminary study originated from equine patients with a variable age, wound history and wound age. Performing a follow-up study on a much larger patient cohort would make it possible to investigate if these parameters have an effect on EGT. It would also be interesting to sample EGT wounds at different time points to see how EGT develops over time. Our results showed a similar abundant number of macrophages in the early phase of physiological healing (6 days post-injury) and EGT, stressing the importance of these cells in equine wound healing and EGT formation. EGT could be very useful in future research to study the inflammatory phases and the role of different pro-fibrotic (M2) macrophages (sub)types in wound healing.


There is a rise in the use and abuse of nitrous oxide (N₂O) as a recreational drug. In spite of the laws enforced internationally, it remains readily available and is an inexpensive mode of recreational drug. Commonly known as the ‘laughing gas’, its use as a euphoric agent is on the rise. Subsequently, the side effects are also coming to light, associated with medical, financial and social implications. It is not detected in routine drug testing. The key differential in an acute setting is often confused with the query for Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy and malabsorption syndromes associated with vitamin B12 and folate deficiencies. This is a case report of a 21-year-old male who presented to the hospital with concerns for weakness and tingling sensations in his extremities accompanied by an inability to bear weight, which he suggested to seem to be worsening over a period of three weeks. His blood tests suggest mild deficiencies of vitamin B12 and folate levels, and MRI revealed subacute combined degeneration of the cervical cord from C2 to C6. The radiologist advised to rule out B12 deficiency and the use of nitrous oxide as a recreational drug. This led to the discussion with the patient, during which he admitted to the use of nitrous oxide. Prompt management with B12 injections intramuscularly every two to three days a week for 11 days followed by folate supplements and monthly B12 injections were advised. He was also reviewed by the physiotherapy teams, and the patient agreed to not use nitrous oxide in the future as a recreational drug. The recreational abuse of nitrous oxide (N₂O) or laughing gas continues to increase in the community. It is a cheaper, euphoria-inducing inhalant agent undetectable on routine drug screening. Although previously thought to be safe, it is now known that nitrous oxide has adverse effects on multiple organ systems, including neurological, immunological and haematological systems . NitrousWe present a case of a 21-year-old male who has a history of inhalational abuse of nitrous oxide and presented inability to get out of the bed with brisk appearance of symptoms of subacute combined degeneration of the spinal cord one day after his shoulder arthroplasty surgery.A 21-year-old male presented to the emergency department and was referred to the medical team on call for concerns of three weeks history of progressively worsening weakness in his arms and legs, inability to stand on his feet, and sensations of tingling in all of his extremities. His past history included COVID-19 AstraZeneca (ChAdOx1 S (recombinant)) vaccine nine months ago, followed by the second dose three months ago. Additionally, he had a remote history of epilepsy stable while on lamotrigine. On further inquiry, he had mild symptoms of tingling in both upper and lower limbs and underwent right shoulder arthroplasty for his history of recurrent right shoulder dislocation. He was discharged home the same day post-surgery. The very next day, he was unable to get out of the bed by himself and decided to come to the hospital for further review. Further discussions with the anaesthesia department revealed that nitrous oxide was not used during the orthopaedic management of the right shoulder dislocation. On examination, he was afebrile and normotensive. Chest, cardiovascular and abdominal examinations were inconclusive. Neurological examination revealed normal reflexes in the upper limbs with brisk knee reflexes and positive Babinski in both lower limbs. His lower limb position and vibration sensations were impaired, along with impaired sensation to touch that normalised above the knees bilaterally. Motor examination showed power 4/5 bilaterally in the upper limbs and 3/5 in the lower limbs. Romberg was positive. The cerebellar examination was normal. MRI of his cervical spine with T1- and T2-weighted images showed sagittal T1 and T2 and STIR sequences. There was a long segment cord signal abnormality involving the dorsal columns with low T1 and high T2 signals extending from C2 to C6 level. Vertebral body heights and intervertebral disc spaces were preserved. There was no focal marrow signal abnormality, nor there was any evidence of spinal canal stenosis , nutritional/metabolic deficiencies  and substance abuse . On further discussions with the patient, he admitted to using approximately 40-120 whipped cream chargers weekly. He underwent thorough blood investigations. The results suggested functional vitamin B12 deficiency with slightly low levels of vitamin B12 and high levels of methylmalonic acid and plasma homocysteine levels  antibodies, syphilis, complete viral hepatitis, HIV screening, and cerebrospinal fluid analysis including viral PCR studies were all completely normal. His lamotrigine levels in serum were also normal. A diagnosis of nitrous oxide-related subacute combined degeneration of the cord was made. Prompt management with B12 injections intramuscularly every two to three days a week for 11 days followed by folate supplements and monthly B12 injections were advised. He was also reviewed by the physiotherapy teams and underwent graded exercises to improve mobility. The patient agreed not to use nitrous oxide in the future as a recreational drug.2O) has long been used as an anaesthetic, commonly used to induce general anaesthesia towards general and other surgeries. However, it is also commonly used for recreational purposes especially in the younger age group [Nitrous oxide (Nge group . It has ge group . Nitrousge group .2O abuse appears to be mediated by B12 deficiency, which is functional, resulting in decreased normal myelin synthesis. A metabolite of cobalamin, namely, 5-deoxyadenosylcobalamin, a coenzyme catalysing the conversion of methylmalonyl CoA to succinyl CoA, which subsequently enters the citric acid cycle, is compromised. Nitrous oxide oxidises the cobalt ion of vitamin B12, which irreversibly inactivates cobalamin, which is unable to function as a coenzyme. This results in the accumulation of methylmalonyl CoA, which enters into the lipid pathway and results in the incorporation of abnormal fatty acids into neuronal lipids. The outcome is subacute combined degeneration of the spinal cord, more appropriately suggesting the decreased myelination of the lateral and posterior columns of the spinal cord [The mode of subacute combined degeneration in the setting of Nnal cord ,10.In a separate pathway, nitrous oxide neurotoxicity is mediated through nitrous oxide binding to and inactivating cobalamin, reducing cobalamin’s effect as a cofactor in the conversion of homocysteine to methionine . Since mSubacute combined degeneration of the spinal cord usually presents with ascending paraesthesia, weakness, ataxia, and loss of sphincter control most commonly reported with vitamin B12 deficiency. MRI shows symmetric increased T2 signal in the posterior and lateral columns of the cervical and thoracic spinal cord with inverted ‘V’ sign .Although methionine supplementation has some evidence of efficacy, its use is hampered by supply and availability at times . DespiteIn a patient presenting with symptoms and signs of vitamin B12 deficiency along with low levels of vitamin B12 and folate levels, it is important to take a thorough history. In the case of low or low normal levels of vitamin B12, it is important to have plasma homocysteine and methylmalonic acid levels to confirm the diagnosis. A prompt vitamin B12 injection along with physiotherapy support is vital towards the clinical recovery albeit with residual symptoms.