Accessory proteins are a key feature that distinguishes primate immunodeficiency viruses such as human immunodeficiency virus type I (HIV-1) from other retroviruses. A prime example is the virion infectivity factor, Vif, which hijacks a cellular co-transcription factor (CBF-β) to recruit a ubiquitin ligase complex (CRL5) to bind and degrade antiviral APOBEC3 enzymes including APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H). Although APOBEC3 antagonism is essential for viral pathogenesis, and a more than sufficient functional justification for Vif’s evolution, most viral proteins have evolved multiple functions. Indeed, Vif has long been known to trigger cell cycle arrest and recent studies have shed light on the underlying molecular mechanism. Vif accomplishes this function using the same CBF-β/CRL5 ubiquitin ligase complex to degrade a family of PPP2R5 phospho-regulatory proteins. These advances have helped usher in a new era of accessory protein research and fresh opportunities for drug development. AIDS argnancies . Soon afgnancies .All retroviruses encode Gag, Pol, and Env polyproteins that are processed by viral and cellular proteases to release individual proteins during nascent particle assembly. Gag encodes structural proteins matrix (MA), capsid (CA), and nucleocapsid (NC), Pol encodes the viral enzymes protease (PR), reverse-transcriptase (RT), and integrase (IN), and Env encodes the surface glycoprotein GP160, which is processed into the receptor-binding GP120 and transmembrane GP41 domains by a cellular furin-like protease . Lentiviin vitro; however, in vivo they are strongly maintained during natural infections. For example, in vitro, Vif, Vpr, Vpu, and Nef exert functions in a cell-type dependent manner, where these genes are dispensable for producing infectious viral particles in some T cell and myeloid cell lines but not others . However, in physiologically relevant primary CD4+ T cells and monocytes, loss of these genes results in severe impairment of HIV-1 replication , immunomodulatory accessory genes are frequently deleted, or dispensable, for viral replication lication . To furt T cells .+ T cells and macrophages, and some immortalized T cell lines (CEM and H9), results in the production of almost no infectious virus (The underlying mechanism of how Vif enhanced HIV-1 pathogenesis remained elusive for many years following its identification. The key to uncovering Vif’s function originated from observations that in some adherent (HeLa and HEK293T) and T cell lines (CEM-SS and SupT1) Vif is dispensable for producing infectious virus. In contrast, loss of Vif in physiologically relevant primary CD4us virus . Comprehus virus .APOBEC3G (A3G) belongs to a family of cytosine deaminase enzymes that converts cytosine to uracil (C-to-U) in single-stranded DNA . DNA C-tbona-fide co-factor required for Vif expression and counteraction of APOBEC3 restriction factors in vivo, and that it is an integral part of the complex as evidenced by a high-resolution structure  interact with four restrictive APOBEC3 enzymes , recruit CBF-β, nucleate the formation of an E3-ubiquitin ligase complex, and induce G2/M cell cycle arrest (discussed in further detail below)? Remarkably, it has become apparent that each Vif-substrate pairwise combination utilizes largely distinct, and genetically separable, interaction surfaces . Importa40YRHHY44 segment being unique to A3G recognition  . Using hInterestingly, the PPP2R5 surface recognized by Vif overlaps with the substrate binding pocket used by PPP2R5s to bind cellular substrates . CrystalThe close proximity and partial overlap between these interfaces raised the question of whether Vif and cellular substrates compete for binding. To test this model, we utilized a well-characterized peptide inhibitor that contains the LxxIxE motif recognized by PPP2R5s to determine if Vif-induced degradation could be blocked. Co-transfection of a plasmid expressing four tandem copies of this LxxIxE peptide caused a dose-dependent inhibition of Vif-mediated degradation of PPP2R5A . HoweverThe discovery of PPP2R5 substrates was a major step forward in understanding Vif-induced cell cycle arrest. However, the regulatory checkpoints altered downstream of PPP2R5 degradation remain unknown. PP2A/PPP2R5 complexes have been shown to regulate entrance and exit of the G2-to-M phase transition at multiple different checkpoints. Below, we discuss these checkpoints in the context of previous observations regarding Vif-induced G2/M arrest and postulate a model in which antagonism of discrete PP2A/PPP2R5 complexes would lead to simultaneous inhibition of multiple checkpoints.First, arguably the most critical event for mitotic entry is CDC25-mediated activation of CDK1, which allows for nuclear translocation of the CDK1-CyclinB complex , top. AcSecond, PP2A/PPP2R5 complexes also control G2-to-M progression through spatiotemporal regulation of the Aurora kinases. Aurora kinases are involved in numerous G2-to-M checkpoint controls including spindle assembly, microtubule-kinetochore attachment, and cytokinesis . To exitLastly, PP2A/PPP2R5 holoenzymes have been shown to serve as a scaffold for multiple distinct complexes to initiate exit from mitosis. For example, Shugoshin (SGO1) directly binds to a PP2A/PPP2R5 holoenzyme to form a complex that regulates accurate chromosome segregation during mitosis , top. WiIn addition to manipulation of the PP2A/PPP2R5 axis, it has been suggested that Vif can induce G2/M arrest by blocking MDM2-mediated ubiquitination and nuclear export of TP53 . This stin vivo, the importance of PPP2R5 degradation remains to be established. However, at least three key observations have been made that imply that alteration of the cell cycle may be beneficial for HIV-1 pathogenesis.Now, Vif clearly has two distinct sets of cellular substrates, APOBEC3 enzymes and PPP2R5 phospho-regulators . While APOBEC3 counteraction has been shown to be essential for viral infectivity and pathogenesis First, HIV-1 Vpr also potently induces G2/M arrest through a mechanism that is clearly distinct from that of Vif. Vpr activates the ATR DNA damage response pathway by inducing proteasomal degradation of diverse cellular substrates using a Cullin 4 (CUL4), DNA damage-binding protein 1 (DDB1), and DDB1-CUL4-associated factor (DCAF1) E3-ubiquitin ligase complex . While VAt least two phenotypes have been associated with Vpr-induced arrest that may enhance HIV-1 pathogenicity. First, degradation of MUS81 and EME1 by Vpr leads to G2/M arrest and prematurely activates the SLX4 complex, which suppresses spontaneous and HIV-1-mediated induction of type I interferon responses and may contribute to immune evasion . Second,+ T cells , human polyoma virus, infectious bronchitis virus, simian virus 40, and adenovirus ]. IntereDS drafted the manuscript. DS and RH edited the manuscript. Both authors contributed to the article and approved the submitted version.RH is a co-founder, shareholder, and consultant of ApoGen Biotechnologies Inc. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rotaviruses (RVs) are recognized as a major cause of acute gastroenteritis (AGE) in infants and young children worldwide. Here we summarize the virology, disease burden, prevalence, distribution of genotypes and seasonality of RVs, and the current status of RV vaccination in Southeast Asia  from 2008 to 2018.Rotavirus infection in Children in Southeast Asia countries was assessed using data from Pubmed and Google Scholars. Most countries in Southeast Asia have not yet introduced national RV vaccination programs. We exclude Brunei Darussalam, and Timor Leste because there were no eligible studies identified during that time.According to the 2008–2018 RV surveillance data for Southeast Asia, 40.78% of all diarrheal disease in children were caused by RV infection, which is still a major cause of morbidity and mortality in children under 5 years old in Southeast Asia. Mortality was inversely related to socioeconomic status. The most predominant genotype distribution of RV changed from G1P[8] and G2P[4] into the rare and unusual genotypes G3P[8], G8P[8], and G9P[8]. Although the predominat strain has changed, but the seasonality of RV infection remains unchanged. One of the best strategies for decreasing the global burden of the disease is the development and implementation of effective vaccines.The most predominant genotype distribution of RV was changed time by time. Rotavirus vaccine is highly cost effective in Southeast Asian countries because the ratio between cost per disability-adjusted life years (DALY) averted and gross domestic product (GDP) per capita is less than one. These data are important for healthcare practitioners and officials to make appropriate policies and recommendations about RV vaccination. Several uncommon RV genotypes such as G2P[8], G8P[6], G5P[19], G9P[4], G9P[6], and G1P7[5] were identified in the surveillance data. The presence of such diversity among RV isolates provides insight into the evolution of these strains, which can arise due to point mutations, genetic rearrangements, reassortment events, and interspecies transmission , 81. CirDifferences in the predominance of RV genotypes and newly emerging strains were identified over the surveillance period in Cambodia. The G1P[8] genotype was predominant in 2010, 2011, and 2012 , whereas genotype G2P[4] predominated between 2013 (54%) and 2014 (44%). The previously uncommon strain G8P[8] also emerged in 2014 (21%). The proportion of G8P[8] genotype detections increased further in 2015 (41%), in conjunction with the emergence of G9P[8] (10%). By 2016, the detection of genotype G9P[8] had increased to 18%, and G3P[8] became the most prevalent genotype (responsible for 63% of detections) .In Indonesia, the surveillance results demonstrated a changing trend for the most prevalent genotype, from G1P[8] in 2009–2013 to G3P[8] in 2014–2018. From 2009– to 2013, G1P[8] was the most prevalent genotype circulating, which accounted for 38, 73.7, 67.5, 85.7, and 60.1% each year, respectively. G3P[8] became the most predominant strain in 2013, and this continued to 2015, accounting for 49,7, 82.5%, and 84,4%, respectively , 55–57. During the 7-year surveillance period in Lao PDR, the most predominant genotypes identified by year were G2P[4] (40%) and G1P[8] (32%) in 2009; G2P[4] (28%) and G9P[8] (26%) in 2010; G3P[8] (61%) and G1P[8] (36%) in 2011; G3P[8] (81%) in 2012; G1P[8] (57%) and G2P[4] (34%) in 2013; G2P[4] (81%) in 2014; and G1P[8] (96%) in 2015 .From 2008 to 2010, the most common genotype in Malaysia was G1P[8] (82%). Other genotypes identified were G2P[4] (7.6%) and G9P[8] (6.3%). Approximately 4% of the samples were either mixed or untypeable  . A 2006 In Myanmar, the most common strains in 2009 were G1P [8] (28.3%) and G12P [8] (28.3%). G12P [8] was detected from 2009 to 2012, ranging from 28.3% in 2009 to 70% in 2011. G2P[4] became the most predominant strain in 2012–2013, followed by G1P[8] in 2013–2014. G9P[8] comprised only 1% of the RV strains in 2011 and increased to 97.5% in 2014. While in 2015, the majority (90%) of RV strains comprised G9P[8] (54%) and G3P[8] (36%). G9P[8] emerged in Myanmar in 2011 and was the most common strain in 2014 and 2015 , 85.In the Philippines, 1949 (98.5%) RVA-positive stool specimens were successfully typed. The most common genotypes identified were G1P[8] (60.3%), G2P[4] (28.1%), and G9P[8] (5.7%). The frequencies of RVGE cases due to G1P[8] were similar in 2013 (72.0%) and 2014 (75.1%), but it decreased to 44.7% in 2015. Likewise, the frequencies of cases due to G2P[4] were similar in 2013 (17.1%) and 2014 (14.1%) but increased to 42.9% in 2015. The proportion of RVGE cases caused by G9P[8] did not change appreciably from 2013 (5.9%) to 2014 (6.9%) or 2015 (4.6%). Mixed genotypes, unusual strains, and animal strains were detected in specimens from 38 (1.9%), 22 (1.1%), and 10 (0.5%) children with RVGE,. Rare and unusual genotype combinations identified include three G1P[4], eight G2P[8], seven G8P [6], two G8P[8], and two G9P[4] strains. Also, animal strains were detected in specimens from 10 children, including 1 G3P[9] feline-like and nine G4P[6] porcine-like strains .The predominant strain observed in Singapore was G1P[8] , while G9P[8]  was the second most common type observed among children in Singapore , 66.In Thailand, G2P[4] was the most common genotype in 2008 . G1P[8] was the predominant genotype in 2009, 2010, 2012, 2013, and 2014, accounting for 47.3, 35.8, and 63.9%, 60.4, and 56.2%, respectively. In 2011 (68.7%), 2015 (47.3%), and 2016 (89.8%), most RV strains were G3P[8]. Uncommon genotypes found were G1P7[5], G5P[19], G9P[4], and G9P[6], adding to the existing list of uncommon genotypes reported to circulate in Thailand , 73–76.Based on RV diarrhea data from four sentinel hospitals in Vietnam from 2012 to 2015, G1P[8] was the most prevalent strain during 2012 and 2013, accounting for 80 and 82% of total genotyped samples, respectively. G2P[4] was found in 5% of samples in 2012 and 9% in 2013. In 2014 and 2015, the proportion of RVGE caused by G2P[4] increased to 36 and 28%, respectively. G8P[8] was not detected in 2012 and 2013, it accounted for only 1% of specimens in 2014, and it became predominant (31%) in 2015 .Meteorological conditions have an indirect yet important impact on the epidemiology of human rotavirus infection. Weather-related low indoor relative humidity and indoor crowding may be key factors in the epidemiology of rotavirus disease. Hospitalizations for rotavirus gastroenteritis tended to be more common after a cold or dry month than after a warm or wet corresponding calendar month .The temporal trend of Cambodian RV infection shows substantial year-round transmission with prominent peaks during colder, dry months. Peaks typically occurred between November and May . In IndoThe seasonal pattern in RV varies by climatic region and is also associated with local weather. A reduction in RV rates was associated with increased temperature and precipitation . There iOne of the best strategies for decreasing the global burden of disease is the development and implementation of effective vaccines . RV is tThe Global Alliance for Vaccines and Immunizations (GAVI), also known as the Vaccine Alliance, actively supports RV vaccination by subsidizing the cost in eligible countries. In LMIC, the budget impact is an important criterion for funding new interventions, particularly for large public health investments such as new vaccines. By the end of 2018, GAVI had funded RV vaccine introductions in 45 countries .Research to develop a safe, effective RV vaccine began in the mid-1970s when investigators demonstrated that previous infection with animal RV strains protected laboratory animals from experimental infection with human RVs . The firDue to the past association of intussusception and the earlier RV vaccine, large safety studies were performed both for RV1 and RV5 before market authorization . RotarixAt the end of 2018 there are four globally available WHO-prequalified oral vaccines  , one rotTwo RV vaccines, Rotarix® and RotaTeq®, have been developed by Glaxo Smith Kline and Merck, respectively. Rotarix® is a live attenuated monovalent vaccine derived from the most common human RV strain, G1P[8]. RotaTeq® is a live attenuated pentavalent vaccine containing mono-reassortant strains with genes encoding the human G1, G2, G3, G4, and P[8] protein in the genetic background of a bovine RV strains , 100. ThRotavac®  is a monovalent human-bovine RV vaccine. The vaccine consists of the 116E RV strain, which is a naturally occurring reassortant strain G9P[11], containing 1 bovine RV gene P[11] and 10 human RV genes . RotavacThe Lanzhou Institute of Biological Products manufactures the Lanzhou Lamb RV vaccine (LLR). It is a monovalent lamb vaccine strain G10P[12], attenuated by cell passage , and wasRotavin-M1 vaccine is manufactured by the Center for Research and Production of Vaccines and Biologicals and was licensed for use in Vietnam in 2012. The vaccine was derived from an attenuated strain, G1P[8], isolated from a Vietnamese child. A clinical trial found the vaccine to be safe and immunogenic in Vietnamese infants .Another candidate RV vaccine, RV3-BB, was developed from a neonatal strain G3P[6] identified in Australia, with ongoing early clinical studies conducted in New Zealand and now underway in Indonesia. It was also successfully implemented for vaccination of neonates . Table 4Intussusception, neutralizing antibodies present in breast milk, as well as the lower vaccine effectiveness in less developed settings has stimulated interest in an alternative, parenteral approach to immunization –126. TheThree types of animal models have been used to evaluate protective efficacy of VLPs: infection models (adult mice and rabbits), disease models (gnotobiotic piglets), and models evaluating passive protection  . GnotobiRV vaccination does not completely protect young children against infection, but it reduce the severity of RVGE . RV vaccAdditionally, RV vaccination confers herd protection among infants and children under 5 years old who had not been vaccinated –146. In Many countries in Southeast Asia have not implemented national RV vaccination programs including Cambodia, Indonesia, Lao PDR, Malaysia, Myanmar, Singapore, Thailand and Vietnam. One reason is because of uncertainties regarding the cost-effectiveness of incorporating RV vaccination into the NIP. In addition, the vaccine’s decreased efficacy in LIC settings has discouraged its introduction. Prevention of diarrhea in these countries has focused on patient treatment and the management of water quality, sanitation, and hygiene .In Indonesia, the RV vaccine has been commercially available since 2011. Indonesia’s national vaccine manufacturer, PT. Bio Farma, Bandung, is developing an RV vaccine using G3P[6] strain in collaboration with the Murdoch Children’s Research Institute in Melbourne, Australia . Bio FarCurrently Rotarix® and RotaTeq® are commercially available in Malaysia through private health providers . HoweverIn Myanmar and Lao PDR where individual incomes are relatively low, international assistance will support for the RV vaccine introduction in 2020. The total amount of GAVI support for Myanmar is $4,088,000 . Lao PDRIn July 2012, the Philippines became the first Asian country to introduce RV vaccines into its NIP. The Philippines has initially focus on immunizing children living in the poorest communities, which have the highest child morbidity and mortality rates from the diarrheal disease . The tarTwo live-attenuated, orally administrable RV vaccines Rotarix® and RotaTeq® were licensed in Singapore in October 2005 and July 2007, respectively . To dateThe National Vaccine Committee of Thailand considered the introduction of an RV vaccine in 2010 in Sukhothai and Petchabun. Sukhothai province began a routine immunization program with an RV vaccine in October 2011. Evaluation of the first introduction was completed in 2017 and concluded that RV vaccine was highly effective in preventing diarrheal hospitalizations and conferring herd protection among older children who had not been vaccinated . TherefoVietnam is located in a region of high RV infection incidence and eligible for financial support to introduce vaccines into the expanded program of immunization (EPI) from the GAVI . In 2012In LMICs, understanding the short- and long-term impact of intervention adoption on national budgets is critical for ensuring program sustainability . Both buFor GAVI non-eligible countries, the price for Rotarix® is $2.49–7.27, and for RotaTeq® is $3.65–5.09 . For GAVWHO-CHOICE (CHOosing Interventions that are Cost-Effective) uses the GDP as an indicator to develop the following widely referenced categories of cost-effectiveness. Disability-adjusted life years (DALYs) averted is a widely used indicator that allows easy comparison with a ‘no vaccination’ strategy and with others public health interventions. The ratio between costs per DALY averted and GDP per capita less than one is defined as highly cost effective. The ratio between 1 to < 3 and ≥ 3 are defined as cost effective and not cost effective, respectively . The lowAccording to 2008–2018 RV surveillance data for Southeast Asia, 40.78% of all diarrheal cases were causes by RV. Acute diarrheal disease caused by RV is still a major cause of morbidity and mortality in children under 5 years old in Southeast Asia. Among all assessed countries, the most predominant genotype distribution of RV changed from G1P[8] and G2P[4] into the rare and unusual genotypes G3P[8], G8P[8], and G9P[8]. Although the predominant RV strain has been changed, but the seasonality of RV infection remains unchanged. Continuous surveillance is necessary to determine whether they are regional genotype differences. Epidemiological data on RV prevalence will greatly facilitate vaccine development. In the mean time, the development of new vaccines will be needed if RVs are able to evade current vaccine immunity. More effective vaccines may also further decrease RV infection in children in LIC and LMIC, where currently available vaccines provide moderate efficacy. RV vaccine efficacy is affected by individual factors and external factors. Although most countries in Southeast Asia have not yet introduced national RV vaccination programs, such introduction is projected to be highly cost-effective because the ratio between costs per DALY averted and GDP per capita it is less than one.


FADS1 (encoding D5D) are in LD with FADS2 (encoding D6D) variants. Our univariable MRs revealed risk-increasing total effects of both, D6D and D5D on type 2 diabetes (T2DM) risk; and risk-increasing total effect of D6D on risk of coronary artery disease (CAD). The multivariable MR approach could not unambiguously allocate a direct causal effect to either of the individual desaturases. Our results suggest that D6D is causally linked to cardiometabolic risk, which is likely due to downstream production of fatty acids and products resulting from high D6D activity. For D5D, we found indication for causal effects on T2DM and CAD, which could, however, still be confounded by LD.Estimated Δ5-desaturase (D5D) and Δ6-desaturase (D6D) are key enzymes in metabolism of polyunsaturated fatty acids (PUFA) and have been associated with cardiometabolic risk; however, causality needs to be clarified. We applied two-sample Mendelian randomization (MR) approach using a representative sub-cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC)–Potsdam Study and public data from DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) and Coronary ARtery DIsease Genome wide Replication and Meta-analysis (CARDIoGRAM) genome-wide association studies (GWAS). Furthermore, we addressed confounding by linkage disequilibrium (LD) as all instruments from  Δ5-Desaturase (D5D) and Δ6-desaturase (D6D) are key enzymes for the synthesis of longer chain poly-unsaturated fatty acids (PUFA) from plant-derived precursor fatty acids linoleic acid (LA) and α-linolenic acid (ALA) see a.Prospective cohort studies used product-to-precursor ratios of fatty acids measured in blood fractions, representing surrogate markers of estimated liver desaturase activity . OverallTo investigate causal relationships between risk factors and disease outcomes, genetic variants can be used as instrumental variables in Mendelian randomization (MR). The principle is based on Mendels’s second law of inheritance, stating that the assignment of alleles is random during meiosis .FADS1 and FADS2 genes . Suggestive significance threshold was defined as p < 1.00 × 10−5. We assumed an additive genetic model, adjusted for age at recruitment and sex. Variants were mapped to Ensembl annotation version 87 (GRCh37)  and(GRCh37) .p < [0.05/169]) instrumental variables and performed clumping according to LD. Therefore, SNPs within a window of 10,000 kb and being in LD as defined by R2 ≥ 0.3 were removed  that were not located within the FADS gene region (with a clumping-threshold of 0.001).We conducted univariable two-sample MR studies using single desaturases as exposures on cardiometabolic outcomes. Effect estimates of the association between genetic instruments and desaturases were obtained from EPIC-Potsdam data and effect estimates of the SNP-T2DM and SNP-CAD associations were used from public summary GWAS data on T2DM  and CAD  removed . The SNP phase 3 . We repeData were harmonized for the direction of effects between exposure and outcome associations and palindromic SNPs were excluded. We used an inverse variance weighted (IVW) meta-analysis of SNP specific Wald ratios (SNP-outcome estimate divided by SNP-exposure estimate) using random effects, to obtain causal estimates for the desaturase activities on T2DM or CAD risk. MR-Egger method  was usedR² < 0.3 leaving ten independent instruments. We obtained the SNP effects on the other desaturase activity and vice versa. Extraction of the outcome GWAS results and data harmonization was conducted as described for the univariable MR. We applied the IVW multivariable MR performing multivariable weighted linear regression with the intercept term set to zero and accounting for the correlation structure among the ten FADS variants. We additionally included genome-wide significant hits (p < 9.36 × 10−9) that were not located within the FADS gene region. As sensitivity analyses, we used multivariable MR-Egger for two-sample summary data ) variants of which after LD clumping seven independent variants (R² < 0.3) remained (Genetic variants within the ated D6D  and D5D ated D6D . For D6Dremained . All of remained .FADS-gene-wide significance (p < [0.05/169]) resulting in 11 independent variants (R² < 0.3)  . All of RP11-372E1.4 locus and an intronic variant (rs11644601) at the RRN3; PDXDC1 locus at chromosome 16  at chromosome 3 within the osome 16 .p = 0.21). The MR-Egger regression showed a positive intercept 0.012 (SE = 0.009) p = 0.185) and OR (1.04 [0.98–1.10])  [95% confidence interval (CI)] = 1.08 [1.06–1.09]) on T2DM . The res8–1.10]) , not supp = 0.0004). The MR-Egger regression showed a positive intercept (0.011 (SE = 0.011), p = 0.306) and no association (OR = 1.00 [0.98–1.05]) (p = 0.36).For D5D, we found a positive total effect (OR = 1.03 [1.01–1.04]) on T2DM . There w8–1.05]) , not ind8–1.05]) . After o8–1.05]) ; howeverWe found evidence for a causal total effect of D6D (OR = 1.06 [1.02–1.11]) on the risk of CAD  with no p < 9.36 × 10−9) for D5D. For both endpoints, there was evidence for heterogeneity between the SNPs . The indication for directional horizontal pleiotropy with regards to CAD remained  from both desaturases were combined leading to ten variants of which all were available within the outcome GWAS . However, the F-statistics were higher . When we accounted for weak instruments, the estimates did not largely change .FADS-instruments: 1.03 [0.97–1.09], p-value = 0.336)  . Exclusi= 0.336) .FADS2 that are in LD with FADS1-SNPs to rule out confounding by LD  and CAD (1.04 [0.98–1.11]), although not significant. Multivariable MR did not indicate direct effects of D5D on risk of T2DM or CAD  cannot be easily separated from those for D5D (FADS1).The D6D catalyzes the desaturation of linoleic acid (LA) that shows inverse association with cardiovascular disease mortality  and cardFADS2 gene that are not in LD with variants from FADS1. This sensitivity analysis revealed stronger total effects of D6D on both T2DM and CAD, which further supports a causal role of D6D. Still, due to the considerable loss of precision in the multivariable MR the allocation of the T2DM-risk augmenting effect of D6D activity exclusively to those PUFAs being substrate or product of D6D activity (LA and GLA) remains relatively uncertain. More likely, downstream formation of PUFAs from GLA mediate an important part of the total effect of D6D.We tried to overcome this problem of confounding by LD for D6D by restricting the selected instruments to those from the FADS1) are largely confounded by LD with FADS2 variants and we were not able to overcome this LD problem by using instruments outside the FADS region. Therefore, our finding of a higher T2DM risk among participants with genetically high D5D activity in univariable MR reflects most likely the genetic linkage with D6D rather than an independent effect.Regarding D5D, alignment of our results from univariable MR with non-genetic observational studies is more complex. D5D, catalyzes the conversion of DGLA to arachidonic acid (AA). Our univariable MR suggested links of higher D5D with higher risk of T2DM. This is in line with a recent MR study showing higher T2DM risk with higher levels of AA . In contFADS-instruments, this result was sensitive to inclusion of genome-wide instruments and outlier exclusions. As pointed out above, it is virtually impossible to account for confounding by LD. Restricting our analysis to variants outside of the FADS-gene region hampered precision of the D5D estimates to an extent that made them uninformative. Hence, we do not consider effect of D5D on CAD risk to be robust and will therefore restrain from causal interpretation.In contrast to T2DM, the observational evidence that links higher estimated D5D activity to lower cardiovascular mortality and CHD risk is rather limited ,5, wherePUFAs play a crucial role in cell membrane fluidity and thereby influence insulin receptor binding affinity and endothelial function  which miFADS region, we used well established genetic candidates from the genic region of the investigated exposures that explain up to ~30 % (rs174555 and D5D) of the variance in the traits (R² ranging from 5 to 10% for D6D and 1 to 30% for D5D). Although we had indication for weak instruments within the multivariable MR, we retrieved comparable estimates when we accounted for that. Besides genetic candidates, we also used genome-wide screen and identified two novel hits for D5D that have not been reported by previous GWAS on estimated desaturase activities [p < 5 × 10−8. Furthermore, we wanted to be more conservative, as we did not replicate those findings in an independent population. Nevertheless, our MR results incorporating those genome-wide hits were comparable to the main analysis when using only FADS variants.By using instruments from the tivities ,9. AlthoFADS1; however, we did not identify genome-wide significant instruments for D6D outside of FADS which could have been used instead.We minimized potential incorrect causal inference due to horizontal pleiotropy by restricting genetic instruments on those that plausibly act directly on the traits as they were located in the coding genes . FurtherOne limitation of our analysis lies in the exclusion of prevalent cases in the EPIC-Potsdam sample which was used for instrument selection and might have weakened the exposure betas. However, compared to the overall study sample this number was small and we aimed to exclude bias from reverse confounding due to the fact that prevalent cases might have different desaturase activity . Also, oIn conclusion, our results suggest that D6D is causally linked to cardiometabolic risk. However, the effect is likely not directly due to its substrate or product fatty acids, but rather due to the downstream production of fatty acids (DGLA and AA) and their products resulting from high D6D activity. For D5D, our MR approach suggests a causal risk-increasing effect for T2DM and CAD risk; however, we were not able to fully rule out confounding by LD. Interventions that affect desaturase activities in PUFA metabolism may have an impact on the pathogenesis of cardiometabolic diseases.


Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein, we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore, the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFNγ production, also Ca2+- and CaM-dependent, was instead not reduced in HNSCC T cells, which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity, but not IFNγ production, and reduced their chemotaxis in the presence of adenosine, thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally, 1-EBIO enhanced INFγ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore, they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers.The limited ability of cytotoxic CD8 Howeverpromised . Ion chapromised .+ channels, the voltage-dependent Kv1.3 and the Ca2+-activated KCa3.1, regulate the electrochemical driving force for Ca2+ influx that is necessary for T cell effector functions , which in turn activates cAMP and PKA and, ultimately, inhibits KCa3.1, thus suppressing T cell motility and cytokine release   . The latdulators . Thus, ddulators . However2+  gene expression has been shown to be altered in TIL of breast cancer patients . CaM is 2+ . CaM bin2+ . CaM is t manner . This pr changes . When in to open . While i to open . In thisPeripheral blood was obtained from 19 de-identified HNSCC patients in the age range of 47 to 84. The eligibility criteria for patient inclusion in the study were a positive diagnosis for HNSCC confirmed by tissue biopsy and no administration of radiation or chemotherapy before the time of drawing the blood  were obtained from Gibco (ThermoFisher). Collagen I, rat tail was obtained from Corning Inc. CXCL12 was obtained from R&D Systems. Stock solution of adenosine was prepared in sterile water. Stock solution of CXCL12 was prepared in sterile PBS containing 0.1% bovine serum albumin. Stock solutions of 1-EBIO and thapsigargin were prepared in dimethyl sulfoxide and used at 0.1% dilution.+ T cells were isolated from PBMCs by negative selection using the EasySep Human CD8+ T Cell Enrichment Kit (STEMCELL Technologies Inc.). The CD8+ T cells were maintained in RPMI 1640 medium supplemented with 10% human serum, penicillin (200 U/ml), streptomycin (200 μg/ml), 1 mM L-glutamine, and 10 mM Hepes. For all experiments, cells were activated in a cell culture dish pre-coated with mouse anti-human CD3 antibody (10 μg/ml) (BioLegend) and mouse anti-human CD28 antibody (10 μg/ml) (BioLegend) for 72 to 96 h, except where specified.Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll-Paque density gradient centrifugation , as described previously . CD8+ T + T cells using the E.Z.N.A. total RNA isolation Kit (Omega Bio-tek). 423 ng of RNA was used to synthesize complementary DNA (cDNA) using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (ThermoFisher). Predesigned primers for RT-qPCR were obtained using TaqMan Gene Expression Assays  to detect the expression of CALM1 (assay ID: Hs00300085_s1), CALM2 (assay ID: Hs00830212_s1), CALM3 (assay ID: Hs00968732_g1), 18s rRNA (assay ID: Hs99999901_s1), and GAPDH (assay ID: HS03929097_g1). The RT-qPCR was set up in a 96-well plate by adding 30 ng of cDNA, 1× TaqMan Gene Expression Master Mix , and 1 μl of TaqMan Gene Expression Assay primers. All samples were run in technical replicates of quadruplicates or triplicates, as indicated in the individual figure or table legends. 18s rRNA was used as an internal control. RT-qPCR was cycled in Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems). CT values were measured using StepOne software version 2.1 (Applied Biosystems). CT values for CALM1, CALM2, and CALM3 were normalized against measured CT values for 18s rRNA, and the ΔΔCT values were calculated as described previously  or biotin-conjugated mouse anti-human KCa3.1 antibody  diluted in blocking solution. The background staining of the Biotin conjugated KCa3.1 antibody is shown in 2 X 10Images were analyzed using ImageJ software . Regions of interest (ROI) analysis: Briefly, in each field, the z-stack in the focal plane of the cell membrane  was visualized, and a ROI was drawn around the cell membrane and another ROI was drawn in the cytoplasmic region of individual T cells. The image threshold and background were adjusted and mean fluorescence intensities  for the CaM channels were measured for the membrane and cytoplasmic ROIs. The ratios of CaM fluorescence in the membrane and cytoplasmic ROIs were calculated for individual cells. For the experiments where cells were stained for KCa3.1, fluorescence intensity of KCa3.1 in HNSCC and HD T cells was measured in ROIs drawn around the cell membranes of individual cells.Membrane and intracellular fluorescence measurement analyses of KCa3.1 and CaM were performed by comparing mean gray values (fluorescence intensity)-distance (μm) profiles (line scans) on acquired images of HNSCC and HD cells. The z-stacks in the focal plane of the cell membrane were identified on basis of distinct membrane KCa3.1 fluorescence. Two to three lines were drawn across each cell, and linescan analysis was performed to identify KCa3.1 and CaM expression using the plot profile function on ImageJ. The abscissa corresponding to outermost KCa3.1 fluorescence peaks on the linescan plot defined the cell surface, where the channel was present. Fluorescence intensities  for KCa3.1 and CaM at the cell membrane (the points of KCa3.1 fluorescence peaks) were measured in every linescan analysis performed.In Situ Detection Reagents Orange  as described by us previously  for 1 h, and stained with mouse anti-human KCa3.1  and rabbit anti-human CaM antibodies  diluted in antibody dilution buffer (Millipore Sigma) for 3 h at room temperature. The specificity of the monoclonal rabbit anti-human CaM antibody is shown in PLA experiments were performed using Duolink eviously . ActivatQuantitation of PLA signals in the acquired images was performed using ImageJ. Briefly, the image threshold and background were adjusted for each image and a ROI was drawn around the cell membranes (identified by CD8 staining) and the positive PLA signals (dots) within the ROI were quantified by particle analysis, normalized to the number of cells counted in each field, and presented as the number of dots counted per cell membrane. For three-dimensional visualization of the PLA signal on the membrane, the z-stacks from representative PLA confocal images of T cells from HD and HNSCC individuals acquired as described earlier were reconstructed into 3D using Imaris image analysis software .+ T cells in whole-cell voltage clamp configuration using an AxoPatch 200B Amplifier (Molecular devices). Pipettes were formed from Borosilicate glass  with a P-97 horizontal puller (Sutter Instruments) and had a resistance between 4 and 5 MΩ when filled with intracellular pipette solution consisted of (mM) 145 K-Aspartate, 2 MgCl2, 8.5 CaCl2, 10 EGTA, and 10 HEPES. The pH value was set to 7.2 using TRIS buffer. Unless stated otherwise 1 µM free Ca2+ concentration was maintained inside the pipette solution. The external solution consisted of (mM) 160 NaCl, 4.5 KCl, 1 MgCl2, 10 HEPES; the pH value was set to 7.4 using NaOH. In a subset of experiments we utilized pipette solution with 3 and 10 μM free Ca2+ concentration. Free Calcium concentration was calculated using a program developed by Stanford University. (http://web.stanford.edu/~cpatton/webmaxcS.htm).KCa3.1 and Kv1.3 currents were recorded from activated CD8CALM1 or scr-RNA, the cells expressing GFP fluorescence were evaluated.Data were acquired using pCLAMP 8.0 software (Molecular Devices) through a 16-bit A-D/D-A interface . Data were low pass filtered at 2 kHz and digitalized at 100 kHz. The electrophysiological protocol to record KCa3.1 currents implemented a ramp pulse from −120 to +50 mV from a holding potential of −70 mV every 15 s. The macroscopic conductance (G) of KCa3.1 channels was calculated as a ratio of the liner fraction of macroscopic current slope to the slope of ramp voltage stimulus after subtraction of the leak current . The KCa+ T cells from HD or HNSCC patients were plated on poly-L-lysine coated coverslips, loaded with 1 μM of Fura-2/AM (ThermoFisher) and Fura-2 intensities were measured using InCytIm2 Ca2+ imaging system  as previously described  and lastly, cells were perfused with 0.5 mM Ca2+ solution for 10–15 min. The 0 mM Ca2+ solution contained (in mM): 155 NaCl, 4.5 KCl, 1 MgCl2, 10 HEPES, 10 glucose, 2 EGTA, pH 7.4; while the 0.5 mM Ca2+ solution contained (in mM): 155 NaCl, 4.5 KCl, 2.5 MgCl2, 10 HEPES, 10 glucose, 0.5 CaCl2, pH 7.4. A standard curve was used to correlate the measured ratiometric Fura-2 intensities (340/380 nm ratio) to the intracellular Ca2+ levels, as per the manufacturer's instructions. Change in Ca2+ (ΔCa2+ flux) was calculated as the difference in the measured Fura-2 ratio from its peak value in the presence of 0.5 mM Ca2+ to the baseline measured after addition of thapsigargin.CD8escribed . Briefly® Pre-designed siRNA against CALM1 gene  and scrambled sequence siRNA  were obtained from ThermoFisher.Silencer6 CD8+ T cells that were isolated from HD and activated for 72 h were transfected using the Amaxa™ P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza Cologne GmbH). 100 nM siCALM1 or scr- RNA were cotransfected with 2 μg GFP (pMAX GFP supplied in the Amaxa Nucleofector Kit) using program E0-115 (program: “T cell stimulated”) using a 4D-Nucleofector™ unit (Lonza Cologne GmbH) as per manufacturer's instructions. The transfected cells were incubated overnight at 37° C and further activated for 24 h with anti-CD3 and anti-CD28 antibodies . The efficiency of transfection was ~30% as determined by cells expressing GFP fluorescence. The cells that expressed GFP were considered to be transfected successfully and were used for electrophysiology, flow cytometry, and chemotaxis experiments.1–6 × 10+ T cells (~1 × 106 cells per condition) were stained with Zombie Aqua Fixable Viability Kit (Biolegend) according to manufacturer's instructions, fixed with 4% paraformaldehyde , stained with ATTO 488–conjugated mouse anti-human KCa3.1 extracellular antibody  without permeabilization. For staining with CaM, fixed cells were permeabilized with 0.1% Triton X-100 (MilliporeSigma) and stained with Alexa Fluor 647–conjugated mouse anti-human CaM antibody . To test for T cell activation, CD8+ T cells were stained live with APC-Fire™750–conjugated mouse anti-human anti-CD69 antibody  and then fixed with 1% paraformaldehyde. For intracellular IFNγ staining, PBMC were isolated from HD and were stimulated for 24 h with cell activation cocktail (Biolegend) and were transfected with either siCALM1 or scr-RNA along with GFP as described earlier. Post-transfection, cells were stimulated for 3 h with cell activation cocktail in the presence of brefeldin A (BD Biosciences) in the presence or absence of 1-EBIO. We also isolated PBMC from HD's and HNSCC individuals and stimulated them for 3 h in the presence of brefeldin A in the presence or absence of 1-EBIO. Unstimulated HD and HNSCC PBMC were also incubated in the presence of brefeldin A. All the cells were first stained with Zombie Aqua Fixable Viability Kit according to the manufacturer's instructions, surface stained with Pacific Blue conjugated mouse-anti human anti-CD3 antibody , fixed and permeabilized using BD Cytofix/Cytoperm kit (BD Biosciences). The permeabilized cells were then stained with Brilliant violet 605 conjugated mouse anti-human anti-IFNγ antibody . For all flow cytometry experiments, data were collected on an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo LLC). In experiments performed on T cells transfected with siCALM1 or scr-RNA, cells that were gated on the GFP positive population (indicative of successful transfection) were used for data evaluation.CD86 CD8+ T cells were activated for 72 h and then transfected with either siCALM1 or scr-RNA along with GFP and further activated for 24 h. The cells were then incorporated in a type I rat tail collagen gel (Corning) as per the manufacturer's instructions and added to the central observation chamber of the μ-Slide Chemotaxis. CXCL12 was used as the chemokine for all chemotaxis experiments and 8 μg/ml CXCL12 was added to the migration medium in the reservoir to the left of the observation chamber to generate a chemokine gradient to measure the baseline chemotaxis as described previously  as described previously . Brieflyeviously . To asseeviously . The slieviously .Post hoc testing on ANOVA was done by multiple pairwise comparison procedures using the Holm-Sidak method. Statistical analysis was performed using SigmaPlot 13.0 (Systat Software Inc.). P value of less than or equal to 0.05 was defined as statistically significant.Statistical analyses were performed using Student's t test (paired or unpaired), Mann-Whitney rank sum test , and ANOVA as indicated. + T cells of HNSCC patients. CaM is a small protein of 148 amino acids (~17kD), which is encoded by three genes CALM1, CALM2, and CALM3, and downregulation of either of these genes can result in decreased CaM protein levels , and then, stained post-permeabilization with anti-CaM antibody  and intracellular anti-CaM (after permeabilization) antibodies  in HNSCC T cells . There was no effect of intracellular Ca2+ on the KCa3.1 conductance in HNSCC T cells  while scrambled sequence siRNA (scr-RNA) was used as control. Transfection with siCALM1 decreased CaM expression in activated HD T cells by 30% as compared to scr-RNA-transfected cells (CALM1 reduced KCa3.1 activity by 30% (CALM1 did not significantly decrease KCa3.1 and CD69 (T cell activation marker) protein levels  using the methodology described previously  reversed the functional consequences of  T cells . We cond+ T cells to infiltrate the tumor and function in the TME. Circulating cytotoxic T cells of HNSCC patients display a reduced ability to chemotax in an adenosine-rich tumor-like microenvironment; a limitation arising from a decrease in their KCa3.1 channel activity  and nucleoside diphosphate kinase B (NDPK-B)  . Althoug(NDPK-B) . It is pductance . Further T cells .2+-activated K channels  to LC. HN received grant support from the NIH (T32CA117846T). TW-D was supported by a Clinical and Translational Science Award (CTSA)–awarded KL2 Mentored grant and a grant from CCTST (1UL1TR001425-01).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E1/2) 0.82 at 0.80 V vs. RHE in 0.1 mol/L H2SO4 solution, which is comparable to the commercial Pt/C (Pt 20 wt%) electrocatalyst. The mass activity of the Fe/N/C-S catalyst can reach 45% (12.7 A g−1 at 0.8 V) and 70% (5.3 A g−1 at 0.95 V) of the Pt/C electrocatalyst in acidic and alkaline solutions. As result, ORR activity of PGM-free electrocatalysts measured by the rotating-ring disk electrode method increases in the following order: Fe/N/C<Fe/N/C-S, in both basic and acidic medium. This scientific work offers a facile approach to design and synthesizes efficient heteroatom-doped catalytic materials for electrochemical reactions in energy devices.The exploration of noble metal-free catalysts with efficient electrochemical performance toward oxygen reduction reaction in the acid electrolyte is very important for the development of fuel cells technology. Novel pyrolyzed heteroatom-doped Fe/N/C catalysts have been regarded as the most efficient electrocatalytic materials for ORR due to their tunable electronic structure, and distinctive chemical and physical properties. Herein, nitrogen- and sulfur-doped (Fe/N/C and Fe/N/C-S) electrocatalysts were synthesized using ferric chloride hexahydrate as the Fe precursor, N-rich polymer as N precursor, and Ketjen Black EC-600 (KJ600) as the carbon supports. Among these electrocatalysts, the as prepared S and N-doped Fe/N/C-S reveals the paramount ORR activity with a positive half-wave potential value ( The electrochemical oxygen reduction reaction is the most significant process at cathode in the polymer electrolyte membrane fuel cell (PEMFC) and anion-exchange membrane alkaline fuel cell (AEMFC). The high-cost platinum and other noble metals based electrocatalysts are still commonly used for ORR because of their unique characteristics, such as low over-potential and high current density  and porosity demonstrated best electrochemical performance. The half-wave potential (E1/2) reaches to 944 mV (vs. RHE), that is 34 mV more positive (910 mV vs. RHE) than a commercial Pt/C electrocatalyst and high limiting current density of 5.5 mA cm−2 at 0.43 V in 0.1 M sodium hydroxide solution. While in 0.1 M H2SO4 electrolyte solution, E1/2 potential of the Fe/N/C-S electrocatalyst reaches 820 mV (vs. RHE), which is only 64 mV less than Pt/C electrocatalyst. From the surface morphology analysis, the Sulfur-doped Fe/N/C electrocatalyst looks mesoporous and the Fe particles are preserved through graphitic layers. This electrocatalyst showed high ORR performance, low H2O2% yield, and followed the four-electron path selectivity due to uniform doping of heteroatoms N, S and Fe metal.In this report, we synthesized heteroatom-doped Fe/N/C electrocatalysts with the porous structure for ORR, to especially improve the activity in acidic electrolyte. Among them, the Fe/N/C-S catalyst with uniform potential catalytic active sites, high surface areas (830 m3.6H2O), 99.0%), hydrochloric acid (36%), ethanol (99.7+%), Superpur Sulfuric acid (96.0%), Calcium Hydride (CaH2) and sodium hydroxide (98.0%) were obtained from China Chemical Reagent Corporation Aladin and Macklin. The commercial Pt/C (Pt 20 wt%) and Nafion (5%) were purchased from Alfa Aesar. The carbon black (Ketjenblack EC600J) was bought from Akzo Nobel, Japan. The deionized water (18.2 MΩ) was used during all experimental work.Melamine (99%), Terethalaldehyde (98%), and dimethyl sulphoxide (99%) were received from Aladdin reagents (Shanghai) Co., LTD. Ferric chloride hexahydrate (FeClin-situ coated on KJ600 carbon black (ca. 0.5 g). The synthesized N-doped carbon material (1 g) was mixed with ferric chloride (3 mmol) through C2H5OH solvent by magnetic stirring. The ethanol was evaporated through a vacuum rotary evaporator machine and further dried at 50°C for 10 h. The resulting dried carbon material was pyrolyzed at high temperature 800°C under inert atmosphere (Ar gas) for 1 h with the heating rate of 5°C per min (HT1). The pyrolyzed powder was dispersed in 0.1 M super sulphuric acid by stirring at 80°C for 8 h (HT1-AL). The unstable and inactive impurities were removed by centrifugation and washing with pure H2O several times. The washed product was dried at 85°C for a whole night and then again pyrolyzed (HT2) at 800°C for another 3 h with a heating rate of 5°C min−1 to get the final Fe-N/C electrocatalyst  was mixed with freshly prepared Fe(SCN)3 solution by magnetic stirring and dried at 80°C overnight. The final S-doped Fe-N/C catalyst was obtained after performing a heat treatment and acid leaching process as like to the Fe/N/C electrocatalyst.To synthesize S-doped Fe-N/C, firstly, we prepared fresh Fe(SCN)The surface morphologies and microstructure of electrocatalysts were analyzed by a field emission scanning electron microscope (JEOL JSM-7800F) and a high-resolution transmission electron microscopy (JEM-2100 and X-Max 80), respectively. The X-ray diffraction testing was done by D8 Advance with Cu Kα radiations. The surface chemical composition and active species of the electrocatalytic materials were examined by X-ray photoelectron spectroscopy (XPS) with an Ultra DLD using a monochromic Al X-ray source. The Ar adsorption/desorption isotherms were obtained by a Micromeritics (ASAP 2020) device. The pore size distribution and specific surface areas data were calculated by using the Brunauer-Emmett-Teller theory. The Raman spectra were recorded by using a Renishaw/Invia Reflex spectrometer coupled with a 633 nm laser.The electrochemical testing of electrocatalysts was evaluated by using a CHI-760D bipotentiostat (China) and three electrodes connected to an electrolytic cell at a constant temperature 30°C. The saturated calomel electrode (SCE) and mercury/mercury oxide (Hg/HgO) were used as reference electrodes. A thin graphite strip was used as a counter electrode. A glassy carbon (diameter = 5.61) surrounded by a Pt-metal ring was used as a substrate for the preparation of working electrode.2H5OH, 50 μL 5% Nafion and 0.45 mL H2O). A total 25 μL of the catalyst solution was dropped on the glassy carbon disk electrode after polishing by 0.3 μm Al2O3, and dried at room temperature. For comparison purposes, commercial Pt/C (Pt 20 wt%) electrocatalyst ink was synthesized with a similar method, by dissolving 1 mg in 1 mL C2H5OH solvent. The cyclic voltamograms were measured by potential cycling between 0.2 and 1.2 V vs. RHE with 900 rpm at 10 mV s−1 scan rate. The electrolytic solutions were saturated with O2 gas for 30 min before the measurements and ohmic drop value (iR drop) was compensated. The CV curves also recorded in nitrogen gas saturated electrolyte solution under similar conditions. The current recorded in the O2-saturated electrolyte was revised by the background capacitive current to obtain the ORR current. The mass activity of the electrocatalyst was calculated by dividing the kinetic current (Jk) to the catalyst loading. The Koutecky-Levich's (K-L) equation (Equation 1) applied to calculate the kinetic current.For the preparation of working electrode, the required amount of catalyst (6–10 mg) was ultrasonically dissolved in 1 mL solution 6 and 1 M Sr(NO3)2 solution. The selectivity of the ORR process can be determined through H2O2 percentage by the Equation 3.The collection efficiency of RRDE was experimentally measured to be 0.386 in 5 mM K3C) and other metal impurities after first heat treatment (HT1) in 3C with PDF No. 23-0298 and Fe3O4 with PDF No. 03-0863 in the Fe/N/C-S electrocatalyst. The XRD results proved that the amount of iron is quite higher after the acid leaching and second heat treatment, which indicates that Fe nanoparticles were protected due to N-doped carbon nanoshells in the S-doped electrocatalyst.The structural properties of electrocatalysts were analyzed through X-ray diffraction. The XRD pattern of electrocatalyst showed characteristic reflections of crystalline iron carbide  and G-band (1594 cm−1) confirmed the presence of graphitic carbon atoms structure in catalysts  as compared to the Fe/N/C electrocatalyst. With heteroatoms doping, the Ar adsorption-desorption curves displayed a typical isotherm type-IV and a capillary condensation phenomenon results from a relative pressure value of 0.3–0.8, which indicates the ordered mesoporous nanostructure. As comparison, S-doped electrocatalyst showed more mesoporosity and mesopore volume than Fe/N/C catalyst but less microporosity, as shown in The graphitic carbon content in catalysts was tested via Raman spectroscopy. The characteristic D-  electrocatalyst was examined under similar conditions. For optimizing the synthetic conditions and ORR performance, we firstly determined the ORR activity of the electrocatalysts as a function of the pyrolysis temperature in the range of 700–900°C. The pyrolysis at high temperature played an important role in boosting the ORR activity for non-precious metal-based electrocatalysts . 2 and N2 has been subtracted to obtain the real current value for ORR  in acid electrolyte is significantly improved as compared to the Fe/N/C catalyst. In 0.1 M H2SO4 acid solution, Fe/N/C-S performed the best ORR activity with an E1/2 potential value ca. 820 mV (RHE), which is only 64 mV less than commercial Pt/C catalyst (884 mV vs. RHE). The E1/2 potential of Fe/N/C-S is positively shifted by 30 mV in the acidic electrolyte. The shift of E1/2 shows that the ORR performance in acidic medium is considerably improved due to heteroatoms doping. The low activity of the Fe/N/C electrocatalyst may be due to low density of Fe-Nx and protonation of active sites in acid solution, such as pyridinic N  in The ORR activity of Fe/N/C and S-doped Fe/N/C-S electrocatalysts was examined in alkaline and acidic electrolytes by RRDE technique. 2O2) yield. The ORR selectivity information mostly obtained from the H2O2 percentage. The ORR process includes three possible routes: one is direct four electrons transfer mechanism with high efficiency, and the others two are indirect routes via H2O2 formation as intermediate with low efficiency  at a high potential value between 0.60 to 0.90 V in ne value shows that the ORR on the Fe/N/C-S electrocatalyst followed the 4e-transfer process over O2 molecule in both acidic as well as basic electrolytes, which is owing to the synergistic effects caused by doping of S, N, and Fe atoms.We further investigated the ORR efficiency of the Fe/N/C-S electrocatalyst and its corresponding reaction mechanism by the hydrogen peroxide  and heteroatoms (S and N) in non-noble catalysts can promote electrochemical performance. The Fe/N/C-S electrocatalyst is a promising candidate to replace conventional noble metal-based electrocatalysts in alkaline and acidic solutions.In conclusion, we prepared heteroatoms doped Fe/N/C electrocatalysts through high-temperature pyrolysis. As prepared, the N, S and Fe-doped Fe/N/C catalyst had shown outstanding ORR performances in both alkaline and acidic electrolytes. The main objectives of this study—to improve the ORR activity in acidic electrolyte—were achieved through the doping of heteroatoms. The electrocatalysts have high BET surface area with a mesoporous structure. The best catalyst exhibited high durability, tolerance to methanol, low HAll datasets generated for this study are included in the article/All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


US6 gene encoding viral glycoprotein D (gD2) inserted at four different genomic sites, including the deletion (del) II and del III sites, the CP77 gene locus (MVA_009–MVA_013) and the I8R-G1L intergenic region. Our data indicate that after 35 passages, there was a reduction in gD2 expression from del II, del III and CP77 sites. Sequencing analysis implicated US6 deletion and mutational events as responsible for the loss of gD2 expression. By contrast, 85.9% of recombinant plaques expressed gD2 from the I8R-G1L site, suggesting better accommodation of transgenes in this intergenic region. Thus, the I8R-G1L intergenic region may be more useful for transgene insertion for enhanced stability.The modified vaccinia virus Ankara (MVA), a severely attenuated strain of vaccinia virus, is a promising vector platform for viral-vectored vaccine development because of its attributes of efficient transgene expression and safety profile, among others. Thus, transgene stability in MVA is important to assure immunogenicity and efficacy. The global GC content of the MVA genome is 33%, and GC-rich sequences containing runs of C or G nucleotides have been reported to be less stable with passage of MVA vectors in cells. The production of recombinant MVA vaccines requires a number of expansion steps in cell culture, depending on production scale. We assessed the effect of extensive passage of four recombinant MVA vectors on the stability of the GC-rich herpes simplex type 2 (HSV-2)  C12L gene encoding serine protease inhibitor-1 [C16L/B22R host-range gene [MVA was derived by more than 570 passages of the chorioallantois vaccinia virus Ankara (CVA) in chicken embryo fibroblast (CEF) cells . Genome ibitor-1  and trunnge gene , and thenge gene . Orthopoxviruses, including vaccinia virus, canarypox virus and fowlpox virus, have been used as vectors for the expression of heterologous genes and are promising vaccine vectors for a variety of infectious diseases, as well as therapeutic vectors for the treatment of cancers. Generally, the capacity of vaccinia virus to accommodate large fragments of heterologous DNA and ability to induce high-level expression of transgenes make it an attractive viral vector in vaccine development. In addition, MVA has a high safety profile, including in immunocompromised individuals. Thus, recombinant MVA vectors expressing antigens of the human immunodeficiency virus , hepatitB5R gene encoding an essential protein (B5) of the enveloped virion form of vaccinia virus was unmutated after serial passages of MVA in two non-permissive cell lines, Vero and MRC-5 cells [The stability of a transgene in a virus vector is important to ensure immunogenicity and efficacy. Previously, we showed that the conserved -5 cells . The ins-5 cells ,20,21,22-5 cells . The del-5 cells . While g-5 cells . From thUS6 gene  was inserted at different loci in MVA, including del II and del III regions, as well as the CP77 gene locus and the I8R-G1L intergenic region. The US6 gene and reporter gene expression cassettes were inserted in forward and reverse orientations in CP77 and I8R-G1L sites. These vectors were evaluated for stability after 35 serial passages in DF-1 cells. Our data indicate that US6 was most stable in the I8R-G1L intergenic region, irrespective of orientation of the gene. Deletion of US6 or a combination of mutational changes (base substitutions or insertions) with consequent abrogation of translational initiation and/or induction of frameshift mutations were detected in recombinant plaques that failed to express gD2 after 35 serial passages in cells.The stability of transgenes in MVA following prolonged serial passages in cells has not been thoroughly investigated. Transgenes with stretches of ≥ four G or C nucleotides have been reported to undergo mutational changes when inserted into MVA and passaged in cell culture . MVA hasDF-1 cells, a spontaneously transformed chicken fibroblast cell line (UMNSAH/DF-1 (ATCC # CRL-12203)) and BHK-21 cells (ATCC # CCL-10) were obtained from the American Type Culture Collection  and were grown in Dulbecco’s Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.05 mg/mL gentamicin. MVA, a gift from Dr. Linda Wyatt and Dr. Bernard Moss (NIAID/NIH), and recombinant MVA vectors were prepared and titered in DF-1 cells. CP77 locus in MVA. A 572 bp left-flanking sequence of CP77  was amplified from MVA genomic DNA by PCR, using the primer pair CGGATCCCAAGTTAACTAATAAATAAAAAGT (CM-84) and CCGCTAGCATTGATAAATAAATTTTTTCTAGTGAT (CM-85). The amplicon was ligated with Zero Blunt TOPO vector  and subcloned as a BamHI-NheI fragment into pcDNA3.1(-)  in which a BglII-NheI fragment encompassing the CMV promoter had been removed, to generate pcDNA3-LF. Similarly, a 600 bp right-flanking sequence of CP77 (corresponding to coordinates 9607 to 10206 of ACAM3000) was amplified from MVA genomic DNA with the primer pair, GGATCCCTCATTTTCCAAATAGTCAAACATTTTGA (CM-86) and GCAAGCTTGTTAATAGAACAAAGTAAGAAAATAG (CM-87). The amplicon was cloned into Zero Blunt TOPO vector and subcloned as a BamHI-HindIII fragment into pcDNA3-LF to generate pcDNA3-LF_RF. The primer pair, GCTAGCGCCTTTCATTTTGTTTTTTTCTATGCTA (CM-88) and GATATCAACCACTTTGTACAAGAAAGCTGAA (CM-89) was used to amplify a 2569 bp fragment from the destination vector pLW44attR [NheI-EcoRV fragment into pcDNA3-LF_RF to obtain the destination vector pCAM17. The shuttle vector pCAM17 was constructed to allow for recombination at the LW44attR . The ampI8R-G1L intergenic site in MVA. The left- and right-flanking sequences of CP77 in pCAM17 were substituted with the left- and right-flanking sequences at the I8R-G1L site, respectively. The I8R-G1L left flank was amplified from MVA genomic DNA with the primer pair, GGATCCCTTATATGAAGTCTATACAGCGAATAGA (CM-113) and GCTAGCTTAGTTATTATCTACAGGAACAAATATAGT (CM-114). The 500 bp amplicon (coordinates 57520 to 58019 in ACAM3000) was cloned into Zero Blunt TOPO vector, and subcloned as a BamHI-NheI fragment into pcDNA3.1(-) that had been linearized with BglII and NheI to generate pcDNA3-18LF. Plasmid pCAM17 was digested with AatII and NheI to excise the CP77 left flank sequence. The I8R-G1L left flank was excised from pcDNA3-18LF as an AatII-NheI fragment and ligated with the linearized pCAM17 to generate pCAM17-18LF. The primer pair, GATATCTCAAACTCTAATGACCACATCTTTTTTTAGA (CM-119) and AAGCTTATGATATACAGGCAATTATGGAATCT (CM-120b) was used to amplify the right flank of the I8R-G1L locus. The 500 bp amplicon (corresponding to coordinates 58023 to 58522 in ACAM3000) was cloned into Zero Blunt TOPO vector and subcloned as an EcoRV-HindIII fragment into pCAM17-18LF (in which the pCAM17 right flank sequence had been removed with EcoRV-HindIII) to generate pCAM18. The shuttle vector pCAM18 was constructed to allow for recombination at the CP77 locus and I8R-G1L intergenic region, respectively. For pCAM17R, the primer pair, GCTAGCATCAACAAGTTTGTACAAAAAAGCTGA  and CGAGAAATAATCATAAATAAGCCCATCAACCACTTTGTACAAGAAAGCTGA  was used to amplify the mH5 promoter, eGFP and p11 promoter cassette in reverse orientation from pLW44attR. The primer, GGGCTTATTTATGATTATTTCTCGCTT  was used with GATATCGCCTTTCATTTTGTTTTTTTCTATGCT  to amplify an 847 bp fragment that contained sequences of the p11 promoter, eGFP and mH5 promoter in the reverse orientation. The CM-90/CM-79 and CM-80/CM-91 PCR products were purified from gels and combined as templates for a second-step PCR with primers CM-90 and CM-91 to amplify a 2569 bp product that contains sequences in the order: cassette B Gateway Conversion System, and mH5/eGFP/p11 in the reverse orientation. The second-step PCR product was ligated with Zero Blunt TOPO vector and ccdB survival 2T1 competent Escherichia coli cells  were transformed. Plasmid recovered from transformed cells was digested with NheI and EcoRV and the cassette B/ mH5/eGFP/p11 fragment was subcloned into pcDNA3-LF_RF to obtain pCAM17R. The pCAM18R shuttle vector was generated using the same strategy that was used for pCAM18, i.e., the CP77 left- and right-flanking sequences in pCAM17R were substituted with the left- and right-flanking sequences at the I8R-G1L site to obtain pCAM18R.Plasmids pCAM17R and pCAM18R were designed for the insertion of transgenes in leftward reading frames (Reverse orientation) at the CP77, and I8R-G1L domains, the HSV-2 (strain MS) US6 gene encoding gD2 (GenBank accession # EU445527.1) was amplified from viral genomic DNA with the primers CACCATGGGGCGTTTGACCTCCGGCGT  and CTAGTAAAACAATGGCTGGTGCGA . The amplicon was ligated with pENTR/D-TOPO vector  to generate pENTR-US6. Isolates of pENTR-US6 were sequenced to confirm nucleotide sequence authenticity. The plasmids pLW44-US6, pCAM17-US6 and pCAM18-US6 were generated by LR recombination between pENTR-US6 and pLW44attR, pCAM17 and pCAM18, respectively, using the Gateway LR clonase enzyme mix . pLW44-US6, pCAM17-US6 and pCAM18-US6 (1 µg each) were transfected into DF-1 cells that had been infected for 1 h with MVA at a multiplicity of infection (MOI) of 0.1, using X-tremeGENE HP DNA Transfection Reagent and protocol described by the manufacturer . Recombinant MVA vectors—MVAdel3-gD2, MVA17-gD2 and MVA18-gD2—expressing gD2 from del III, CP77 and I8R-G1L sites, respectively, were isolated by 3 to 5 rounds of plaque purification, expanded in DF-1 cells and partially purified on 36% sucrose cushions as previously described [The recombinant MVA-gD2 (MVAdel2-gD2) vector expressing HSV-2 (strain MS) gD2 from the MVA del II site has been described . For insescribed . US6 in the reverse orientation at CP77 and I8R-G1L domains, the US6 sequence was amplified from HSV-2 (strain MS) genomic DNA with the primer pair, CACCCTAGTAAAACAATGGCTGGTGCGA  and GGCATGGGGCGTTTGACCTCCGGCGT . The PCR product was ligated with pENTR/D-TOPO vector to generate pENTR-US6R, and isolates were sequenced. The plasmids pCAM17-US6R and pCAM18-US6R were generated by LR recombination between pENTR-US6R and pCAM17R and pCAM18R, respectively, using the Gateway LR clonase enzyme mix. Recombinant MVA17-gD2rev and MVA18-gD2rev expressing gD2 from a leftward-reading US6 sequence were generated and purified as described above. For the generation of recombinant MVA vectors with US6 inserted at the four different sites were similar to that of MVA (data not shown). The growth curves of recombinant MVA vectors with Monolayers of DF-1 cells in 6-well tissue culture plates were infected with MVA-gD2 vectors, at an approximate MOI of 0.1. Following virus adsorption for 2 h, infection medium was aspirated, and 1 mL of fresh DMEM growth medium added to each well. Infected cells were incubated for 48 h, followed by three freeze–thaw cycles to release virions. The lysates were clarified by low-speed centrifugation at 1000 rpm for 10 min at 4 °C and stored at −80 °C as the inoculum for the next passage. This process was repeated 35 times for each recombinant MVA vector. MVAdel2-gD2 was also passaged 35 times in BHK-21 cells.In evaluating gD2 expression, DF-1 cells were infected with passage (P)1, P5, P10, P15, P20, P25, P30 and P35 of recombinant MVA vectors at a MOI of 1.0. Cells infected with MVA served as a control. Lysates of infected cells obtained 24 h after infection were tested for gD2 expression by Western blotting as previously described [50)) was extrapolated as the x-axis coordinate corresponding to the intercept of the stained/unstained curves. Immunostaining of MVA plaques expressing gD2 was performed as previously described , with moUS6, and amplicons were ligated with PCR II Zero Blunt TOPO vector , and sequenced with T7 and Sp6 promoter primers, and US6-specific internal primers. The sequencing data were aligned with published sequence data available in the database (GenBank accession # EU445527.1).Monolayers of DF-1 cells were infected at a MOI of 1.0, with MVA or recombinant MVAdel2-gD2 plaque isolates. After a 24-h incubation, infected cells were harvested and collected by refrigerated (4 °C) centrifugation at 1000 rpm for 10 min. The cell pellet was re-suspended in 100 µL of PBS, and total genomic DNA was isolated from cells using the DNeasy blood and tissue kit, and protocol described by the manufacturer . DNA was eluted from the column with 100 µL of ultrapure water, and the eluate was used as template in PCR assay, using the Phusion high-fidelity DNA polymerase, and protocol described by the manufacturer . For PCR assay, the primers CM-63 and CM-62 were used for the amplification of US6 gene in MVA, the gene was inserted into four regions of the MVA genome, including the del II and del III regions, the CP77 locus, and the I8R-G1L intergenic region, by homologous recombination of MVA with plasmid shuttle vectors  deletion located toward the left terminal of the MVA genome, and the del III site carries an approximately 4.7 kbp deletion located toward the right terminal of the genome. These del regions were selected in this study primarily because of the availability of plasmid shuttle vectors for homologous recombination of transgenes into these sites, and the del III site is frequently used as a gene insertion site for candidate MVA-vectored vaccines in preclinical studies. Previously, we described the construction of the MVA vector expressing gD2 from the del II site . For the vectors  were tesRecombinant MVAdel2-gD2 and MVAdel3-gD2 vectors were analyzed for gD2 expression by Western blotting and by immunostaining at intervals of five passages. The former assay allows for a qualitative determination of gD2 expression, while the latter allows for a quantitative determination of the proportion of recombinant plaques expressing gD2. In Western blotting analysis, decreasing levels of gD2 expression from both del II and del III sites were detected with increasing in vitro passage but a similar level of expression of the 14K viral A27 protein was obtained at the different passage levels A,C. Howe50 for MVAdel2-gD2 was determined to be 15.4  are in opposite transcriptional orientations, with I8R being rightward-reading and G1L being leftward-reading. To assess whether the orientation of US6 has any impact on its stability in MVA, the US6 gene was inserted in leftward (reverse) orientation at the CP77 locus and I8R-G1L intergenic region to generate MVA17-gD2rev and MVA18-gD2rev, respectively. A gradual decrease in gD2 expression was observed when MVA17-gD2rev was passaged in cells but without a decrease in A27 expression  plaques were randomly selected, isolated and expanded in cells. In Western blotting, none of the 10 plaque isolates expressed gD2 (not shown). Genomic DNA was extracted from each isolate and used as a PCR template to amplify the US6 gene. US6 was amplifiable from 4 of the 10 plaque isolates. To verify the gD2 expression status of the isolates, two US6 PCRpositive and two US6 PCRnegative isolates were titered on DF-1 cells, and infected cells were immunostained for gD2 expression. None of the isolates had detectable plaques stained with HSV-2 antibody. This set of data suggests that the US6 gene may have been excised from the MVA vector (PCRnegative isolates) or mutated (PCRpositive isolates) during in vitro passage in cells. As described above, more than 50% of recombinant MVA vectors no longer expressed gD2 after 15 to 30 passages when US6 sequences amplified by PCR above were ligated with Zero Blunt TOPO plasmid vector and two plasmid DNA isolates from each were sequenced using T7 promoter and Sp6 promoter primers, along with US6-specific primers. Analyses of the sequence data revealed that each PCRpositive isolate contained at least two combinations of five different mutations, including an A to T substitution at position 1 that altered the AUG (Met) translational initiation codon to UUG (Leu) (CGC to TGC (Arg to Cys) substitution at position 1032, was present in all isolates  . With thisolates . US6 sequence ; a C insertion after a series of six C nucleotide run at coordinates 305 to 310 (found in three isolates); and a C insertion after a series of six C nucleotide run at coordinates 945 to 950 (present in two isolates). Each pair of DNA isolate originating from the same non-gD2 expressing plaque had identical sets of mutations, except for sequences from clone 1, where isolate 1-1 did not contain the ATG to TTG mutation at position 1  CPXV025 in CPXV strain Brighton Red (GenBank # AF482758.2) and ORF CVA017–CVA021 in CVA) and responsible in part for the permissiveness of productive CPXV replication in CHO cells [CP77 is present but fragmented into 4-5 pieces and non-functional. Absence of the CP77 gene product accounts in part for the lack of permissiveness of CHO cells for vaccinia virus replication [CP77 locus in the IHD-J and Western Reserve (WR) strains of vaccinia virus. Passage of IHDJ-luc but not WR-luc in cells resulted in a noticeable reduction in luciferase expression. A loss of gD2 expression was observed with increasing passage of MVA vectors with US6 at the CP77 locus ), it is not a functional translation initiation codon in eukaryotic cells. Thus, recombinant MVA-gD2 plaques carrying this mutation will not be able to initiate the translation of US6 messenger RNA into gD2, accounting in part for the absence of gD2 expression. Wyatt et al. [US6 gene used in this study carries a total of 23 stretches of C nucleotide of ≥ 4 . Four stretches of G nucleotide (comprising three 4 G-runs and a 5 G-run) are also present in US6. However, the base insertions or deletions detected in our study occurred only after six or seven C-runs. Of note, the only stretch of seven C nucleotides is adjacent to a stretch of four G nucleotides, and all mutants contained a C-insertion after the stretch of seven C nucleotides, suggesting that DNA regions with higher numbers of nucleotides in a stretch of Cs are more prone to base insertion or deletion. Thus, important consideration should be given to the nucleotide sequence of a transgene that is to be recombined into MVA. The introduction of silent codon changes to disrupt G- or C-runs [Further gene expression and sequencing analyses indicated that gene deletion and mutational events contributed to the loss of gD2 expression after passage of MVA vectors in cells , includit et al.  reportedr C-runs  or chemiTaken together, our data underscore the need for a thorough assessment of a combination of various factors, including insertion site and the sequence of a transgene, in the design and construction of recombinant MVA-vectored vaccines. Such considerations are paramount for transgene stability, as they could impact the effectiveness of MVA-vectored vaccines. In addition, extensive passage of recombinant MVA vectors is not recommended during the production of MVA-vectored vaccines.


In this work we sequenced phage Xp12 and SP8 genomes and examined Type II restriction of T4gt, T4, Xp12, and SP8 phage DNAs. T4gt, T4, and Xp12 genomes showed resistance to 81.9% (186 out of 227 enzymes tested), 94.3% (214 out of 227 enzymes tested), and 89.9% (196 out of 218 enzymes tested), respectively, commercially available Type II restriction endonucleases (REases). The SP8 genome, however, was resistant to only ∼8.3% of these enzymes (17 out of 204 enzymes tested). SP8 DNA could be further modified by adenine DNA methyltransferases (MTases) such as M.Dam and M.EcoGII as well as a number of cytosine DNA MTases, such as CpG methylase. The 5hmdU base in SP8 DNA was phosphorylated by treatment with a 5hmdU DNA kinase to achieve ∼20% phosphorylated 5hmdU, resulting resistance or partially resistant to more Type II restriction. This work provides a convenient reference for molecular biologists working with modified pyrimidines and using REases. The genomic sequences of phage Xp12 and SP8 lay the foundation for further studies on genetic pathways for 5mC and 5hmdU DNA base modifications and for comparative phage genomics.To counteract host-encoded restriction systems, bacteriophages (phages) incorporate modified bases in their genomes. For example, phages carry in their genomes modified pyrimidines such as 5-hydroxymethyl-cytosine (5hmC) in T4 Escherichia coli (E. coli) strains encoding McrBC and McrA, which are modification-dependent restriction systems that attack 5hmC/5mC-containing DNA (gt is a mutant deficient in DNA α- and β-glucosyltransferases and as a result contains 5-hydroxymethycytosines (5hmC) in its genome (Xanthomonas oryzae) contains 5-methylcytosines replacing all cytosines in its genome  in its genome (Type II restriction and modification (R-M) systems encoded by bacteria and archaebacteria restrict foreign DNA from bacteriophages (phages) and mobile genetic elements . The comning DNA . Phage Ts genome  and thuss genome ,b. The Bs genome .in vitro  and compile a reference list for molecular biologists who use REases to create recombinant DNA. As part of this goal, we sequenced phages Xp12 and SP8 genomes and deposited the sequences in GenBank. In addition, we confirmed the modified base compositions in these four phage genomes by LC-MS. We examined several adenine and cytosine methyltransferase activities on SP8 DNA. We also tested Type II restriction on SP8 DNA after its phosphorylation by treatment with 5hmdU DNA kinase. The results detailed herein comprise a comprehensive reference for the in vitro activity of Type II R-M enzymes on hypermodified DNA.It has been known for many years since the discovery of Type II R-M systems that these modified phage genomes are somewhat resistant to Type II restriction in vitro . But onlin vitro . The goain vitro  on the fgt were kindly provided by Dr. Elisabeth Raleigh (NEB). T4GT7 DNA was provided by Dr. Geoff Wilson (NEB). The bacteriophages supplying the genomic DNAs used in this study were cultured by infection of host cells at early log phase in liquid medium/broth cultured until a significant drop in optical density (OD) occurred indicating lysis of the majority of the cells in the culture. Phage particles were precipitated from centrifugated clarified lysates by addition of PEG8000 to 10% weight-by-volume (w/v) and 1 M NaCl to the phage lysates and collected by centrifugation. Phages were further purified by cesium chloride density gradient centrifugation and dialyzed against three changes of phage buffer . DNA was extracted from the phage by phenol-CHCl3 extraction, and ethanol precipitation . Bacteriophages T4 and T4pitation .REases, MTases, 5hmdU DNA kinase, and Proteinase K were provided by New England Biolabs, Inc., (NEB). NEBcutter v2.1 software  was usedSP8 phage DNA was methylated by treatment with excess DNA MTase and methyl-donor SAM in the recommended buffer for 2 h. After heat inactivation of the MTase (65°C for 30 min), the methylated DNA was digested by cognate or non-cognate REases to evaluate the degree of resistance to restriction.+ 228.1 and [M − H]– 226.2; dG [M + H]+ 268.1 and [M − H]– 266.1; dT [M + H]+ 243.1 and [M − H]– 241.1; dA [M + H]+ 252.1 and [M − H]– 250.1; 5mdC [M + H]+ 242.1 and [M − H]– 240.2; 6mdA [M + H]+ 266.1 and [M − H]– 264.1; 5hmdC [M + H]+ 258.1 and [M − H]– 256.1; α- and β-5hmdC [M + H]+ 420.2 and [M − H]– 418.1. The relative abundance of each nucleoside was determined by dividing the UV absorbance by the corresponding extinction coefficient at 260 nm. To estimate the ratio of phosphorylation of 5-hydroxymethyluridine, kinase-treated SP8 gDNA was digested to nucleotides by treatment with Nuclease P1  overnight at 37°C. Each nucleotide was identified as follows: dCMP [M + H]+ 308.0 and [M − H]– 306.0; 5hmdUMP [M − H]– 337.0; dGMP [M + H]+ 348.0 and [M − H]– 346.0; dAMP [M + H]+ 332.0 and [M − H]– 330.0.Modified or unmodified phage DNA was precipitated in ethanol, dried, and stored at −20°C. DNA samples (5 μg) were digested to nucleosides by treatment with the Nucleoside Digestion Mix  overnight at 37°C. Nucleoside analysis was performed on an Agilent LC/MS System 1200 Series instrument equipped with a G1315D diode array detector and a 6120 Single Quadrupole Mass Detector operating in positive (+ESI) and negative (−ESI) electrospray ionization modes. LC was carried out on a Waters Atlantis T3 column  with a gradient mobile phase consisting of 10 mM aqueous ammonium acetate (pH 4.5) and methanol. MS data acquisition was recorded in total ion chromatogram (TIC) mode. Each nucleoside was identified as follows: dC [M + H]de novo assembled using the HGAP2 algorithm each yielding a single contig with average 200-fold coverage. Open reading frames and some gene assignments were performed by Rapid Annotation of Subsystems Technology (RAST) via web server1  according to manufacturer’s instructions. A total of 5 μg of sheared genomic DNA was used to prepare libraries for Pacific Biosciences (PacBio) Single Molecule Real-Time (SMRT) sequencing on the RSII model sequencer using P6-C4 chemistry and a flow-cell for each phage library. Following sequencing, reads were  server1 . The phaPhage DNAs were extracted from phage particles, purified by the CsCl gradient centrifugation and had their base composition analyzed by the LC-MS as described previously . Table 1gt, T4, and Xp12 genome show 81.9, 94.3, and 89.9% resistance to all Type II REases, respectively  enzyme: 5mC, 5hmC, 5fC (5-formylcytosine), 5caC (5-carboxycytosine) and subsequent removal by the thymine DNA glycosylase (TDG) repair enzyme . Thus, tectively . In addigt, a mutant T4 strain having diminished glucosyltransferase activity, contains 5hmC replacing all C in its genome. Due to the presence of 5hmC, the gDNA was previously reported by others to be resistant to some restriction digestions in vitro , but an extensive list of all commercially available REases is lacking. We purified phage T4gt DNA and analyzed its base composition. T4gt genomic DNA contained ∼83% of 5hmC, and 10% of α-5gmC and 7% of β-5gmC among all cytosine bases . T4gt DNA can also be used as a substrate for modification-dependent endonucleases (see below). Despite the presence of residual 5gmC, we decided to use the T4gt DNA for testing Type II restriction. Examples of restriction digestion are shown in ApoI (RAATTY), AseI (ATTAAT), and a Type IIS enzyme BtsCI (GGATGN2↓) completely digested the DNA. The DNA is nearly resistant to digestion by PflFI (GACN3GTC), and completely resistant to digestion by NaeI (GCCGGC), PstI (CTGCAG), or ScaI (AGTACT). While BccI (CCATC), MlyI (GAGTC), and MboII (GAAGA) partially digested the substrate. Further restriction results are presented in gt was resistant to 81.9% of all Type II restriction enzymes tested here (186 out of 227 enzymes).The DNA of phage T4ne bases . The resgt DNA, it is reasonable to assume that negative restriction on T4gt would be mirrored on T4 DNA. To confirm this hypothesis, we tested twenty enzymes and found that all of them were negative on both T4gt and T4 (data not shown). Next, we selected and tested a subset of REases that could either completely or partially cleave T4gt. Examples of Type II restriction of phage T4 DNA are shown in MseI (TTAA), and NdeI (CATATG) digested T4 DNA completely. But AseI (ATTAAT), SspI (AATATT), and SwaI (ATTTAAAT) did not show complete digestion, even though they all recognize sites containing A/T sequence and they do not overlap with dam methylation site. This partial digestion was reproducible (data not shown) and suggested an indirect inhibitory effect of 5gmC modifications on REases having only A/T nucleotides in their targets. T4 DNA was nearly resistant to HpyCH4III (ACNGT) restriction, and completely resistant to restriction by MboII (GAAGA), NsiI (ATGCAT), or SalI (GTCGAC), all of which containing 2–4 modified cytosines in their target sites. Further Type II restriction data are presented in The base composition of phage T4 DNA was confirmed by LC-MS analysis to carry 60% α-5gmC and 40% β-5gmC . Based o+ Dam+ phage λ DNA (– phage λ DNA displayed a much lower level of 6mA (<0.05%) (Hydroxymethylation-deficient T4 GT7 and λ DNA were used as controls. Only low levels of modified bases 5mC (0.3%) and 6mA (0.5%) were found in phage T4 GT7 . The 5mCge λ DNA . As coul(<0.05%) . Type IIMT664984). The detailed analysis of the Xp12 genome and 5mC modification pathway will be published elsewhere (PW). Examples of Type II RE cleavage of phage Xp12 DNA (5mC) are shown in AfeI (AGCGCT), ApaI (GGGCCC), and ApaLI (GTGCAC). It was partially resistant to Type IIS enzymes MlyI (GAGTC) and MseI (TTAA) (a 3 kb MseI fragment was missing). AhdI, BstNI, KpnI, MboII, and TspA15I completely digested Xp12 DNA, even though their target sites contain 2–5 modified cytosines. It is known that M. AhdI, M. KpnI, and M1. MboII (GGAGG) are N6-adenine MTases and M2. MboII (TCTTC) is an N4-cytosine MTase . The Bacillus phage SP8 contains 100% 5hmdU replacing all T in its genome (ApaLI (GTGCAC) and AseI (ATTAAT), and completely resistant to restriction by BsmI (GAATGC with three 5hmdU), BspMI (ACCTGC), and EcoRI (GAATTC). Five REases  completely digested the substrate DNA. While both EcoRI and EcoRV sites contain four 5hmdU replacing T in SP8 DNA, its sensitivity to restriction was completely different: SP8 DNA was resistant to EcoRI digestion (24 EcoRI sites in the genome) but sensitive to EcoRV restriction. The genomic DNA can be digested by MluCI (AATT). Therefore, it is difficult to predict which REase will completely digest SP8 DNA, even with the presence of up to six 5hmdUs in their target recognition sequences. The SP8 genome was resistant to only a small fraction (∼8.3%) of all Type II restrictions tested here . SP8 DNA, however, was a poor substrate for M. TaqI (TaqI was able to modify λ DNA and rendered the DNA resistant to TaqI restriction (data not shown). M. EcoRI was able to modify λ DNA and protect it from EcoRI restriction. However, in the case of SP8 DNA, no conclusion was derived from the results because the substrate DNA itself was already resistant to EcoRI even before methylation (24 EcoRI sites present in the genome). We have not analyzed other adenine methyltransferase activities on phage SP8 DNA due to the lack of enzyme availability. Overall, phage SP8 DNA can be efficiently methylated by C5 MTases  as one expected. These MTases could modify phage SP8 efficiently and render the DNA resistant to both cognate restriction and overlapping restriction  or M.EcoGII (frequent adenine methyltransferase). The modified DNA became partially resistant to restriction by  M. TaqI . In a cotriction . The metgt, T4, Xp12, and T4GT7 were digested with AbaSI, FspEI, LpnPI, MspJI, and McrBC. gt DNA efficiently. Only AbaSI (a PvuRts1I homolog) was able to digest T4 and T4gt DNA (gt and T4 DNA in ATP/GTP dependent manner (MDREs (Type IIM and Type IV) cleave modified DNA specifically . These eT4gt DNA . T4 DNA t manner .NcoI restriction after the kinase reaction2. We used the 5hmdU DNA kinase to phosphorylate phage SP8 DNA. After the kinase treatment, the DNA was purified by spin column and its base composition was analyzed by LC-MS. 5hmdU DNA kinase can phosphorylate the 5-hydroxymethyl group in the 5hmdU in a sequence-specific manner  making tNcoI, AlwNI, NdeI, BbvCI, BccI, MslI, NlaIII, PvuII, PmlI, NspI, and NsiI, and partially resistant to restriction by EcoRV, XmnI, MlyI, MboI, and Hpy188I , T4 (5gmC), Xp12 (5mC), and SP8 (5hmdU). We found T4gt, T4, and Xp12 genomes are highly resistant to Type II restriction; while SP8 DNA is only modestly resistant to Type II restriction.In this work, we analyzed Type II restriction of modified phage genomes T4A few REases that recognize only A/T target sites partially digested phage T4 and Xp12 DNA. It is not clear whether this observation is an artifact or true indirect effect of base modification on neighboring sequences. In any event, practitioners using REases to digest hypermodified phage genomes should be aware of this partial inhibition.gt DNA efficiently. No undigested phage DNA was detected. It is important to note that LC-MS analysis of T4gt DNA indicated that only 83% of cytosines are modified as 5hmC, with the remaining cytosines found in the form of 5gmC. We have observed a slight variation in phage T4gt base composition (83 to 90% of 5hmC vs. 10 to 17% of 5gmC) across independent batches of phage lysates. The reason for this variation is unknown, but it may be related to suppressor efficiency of different E. coli strains growing T4gt α- and β- glucosyltransferase amber mutants.The MDREs  tested here digested phage T4Phage T4 DNA shows highest resistance to bacteria-encoded Type II restriction. However, bacteria also evolved new restriction systems that can target 5gmC-modified DNA. Examples of these restriction systems include PvuRts1I-like enzymes , GmrSD-lTaqI modified the viral DNA poorly. The cytosine MTases tested here mostly recognize G/C sequence (except M. AluI) and they methylated SP8 DNA efficiently, rendering it resistant to cognate restriction or overlapping restriction. In addition, we tested Type II restriction of phosphorylated (p) SP8 DNA following treatment with 5hmdU DNA kinase. The kinase-treated DNA became more resistant to Type II restriction only if the targets sites contained TG (NG) or TC dinucleotides. A few enzymes (AatII and HinfI) were not affected by base phosphorylation. 5hmdU-containing plasmid or phage DNA could be potentially grown in an engineered E. coli strain to achieve ∼75% maximum incorporation , KpnI (GGTACC), PvuII (CAGCTG), and RsaI (GTAC) endonucleases. Deprotection of the photocaged DNA by UV light treatment (355–425 nm) converted it to 5hmdU-containing DNA which could be cleaved by the REases (in vitro transcription efficiency by E. coli RNA polymerase and restriction by AluI (AGCT) and RsaI (GTAC)  can be incorporated into PCR DNA, which becomes resistant to RsaI restriction. Photochemical release of the protected bases by UV or visible light treatment converted it to 5hmC-containing DNA, which is sensitive to RsaI restriction oxymethyl]-2′-deoxyuridine 5′-e REases . Our worI (GTAC) . AluI sitriction . We founMT664984 and MW001214.Phage Xp12 and SP8 genome sequences have been deposited in GenBank and assigned the accession numbers https://www.ncbi.nlm.nih.gov/genbank/, MT664984 and MW001214.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: KF, S-yX, IC, PW, and ND generated and analyzed experimental data. S-yX, KF, IC, and PW wrote the manuscript. All authors contributed to the article and approved the submitted version.S-yX, IC, ND, and PW are employed by New England Biolabs, Inc. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.