In 2015, foot-and-mouth disease (FMD) virus lineage Ind-2001 was detected for the first time in Southeast Asia. This report contains the first near-complete genome sequence of a viral isolate from this lineage collected from an outbreak in Vietnam. This novel incursion has substantial implications for regional FMD control measures. Picornaviridae family (Foot-and-mouth disease (FMD) is endemic in several countries in Asia and Africa , 2 and ie family . Currenthttp://www.wrlfmd.org/fmd_genotyping/2009/WRLFMD-2009-00005-United%20Arab%20Emirates-O.pdf). However, in 2013 to 2015, several outbreaks caused by sublineage Ind-2001d were reported from Libya, Morocco, Algeria, Tunisia, and the Persian Gulf States  topotype. This lineage has been dominant in the Indian subcontinent since 2008 but is exotic to Southeast Asia . Limited States 58, and itThe virus described herein, O/VIT/16451DLBPP04/2015, was isolated from an epithelial lesion collected from a pig in September 2015 in Ðắk Lắk Province, Vietnam. Acute and rapid onset of vesicular disease was detected in 45 of the 50 pigs present in the affected farm. Virus isolation was achieved by a single passage in BHK cells, as previously described . Viral Rlpro, 2A, 2B, 2C, 3A, 3B, 3C, and 3D). The highest identity with FMDV sequences publicly available is 98%, specifically from viruses collected from the Karnataka and Assam states in India between 2013 and 2014 . FMDV O/VIT/16451DLBPP04/2015 had no polyprotein indels with respect to the Indian sequences.Here, we report the near-complete sequence of the novel isolate FMDV O/VIT/16451DLBPP04/2015. This sequence codes for the complete 6,999-nucleotide (nt) ORF, 71 nucleotides in the 5′ UTR, and 38 nucleotides in the 3′ UTR. The ORF encodes a single polyprotein, which undergoes complex proteolytic processing, ultimately resulting in 4 structural proteins (VP1 to VP4) and 8 nonstructural proteins (http://www.wrlfmd.org/fmd_genotyping/asia.html) further emphasize the importance of vigilance and characterization of novel introductions as they occur.The outbreaks reported in Laos and Vietnam in 2015 were the first incursions of FMDV lineage Ind-2001 ever reported, to our knowledge, in Southeast Asia. Characterization of this virus has substantial implication for control measures, which are normally tailored toward endemic viruses and must be reassessed for new viruses. The regional situation with abundant susceptible hosts and a substantial diversity of cocirculating FMDV serotypes and lineages elevates the concern of the potential emergence of new viruses. Subsequent outbreaks, caused by independent introductions of the same sublineage in Myanmar, Thailand, and Vietnam in 2015 and 2016 (KY657269. The version described in this paper is the first version, KY657269.1.The genome nucleotide sequence of O/VIT/16451DLBPP04/2015 described herein has been deposited in GenBank under the accession no.


Zika virus RNA is frequently detected in the semen of men after Zika virus infection. To learn more about persistence of viruses in genital fluids, we searched PubMed for relevant articles. We found evidence that 27 viruses, across a broad range of virus families, can be found in human semen.  The finding by Atkinson et al. that Zika virus RNA is frequently detected in the semen of men after infection  AND sex* AND Transm*.” Our search revealed that 27 viruses that can result in viremia have been found in human semen . For manGiven these findings, the following questions need to be addressed: which viruses are shed and remain viable in semen, for how long, and at what concentrations? The answers to these questions have implications for risks for sexual transmission and, therefore, embryonic infection, congenital disease, miscarriage, and effects on epidemiologic and transmission models. The presence of virus in the male reproductive tract may increase the risk for acquisition of sexually transmitted infections and may reduce male fertility through spermatogonial stem cell infection or local inflammation. Infection of spermatozoa could result in transmission of virus-induced mutations to subsequent generations, thereby elevating risks for cancer and other disorders. Indeed, when virus has been detected in human semen, the extent to which virus existence and replication occurs within spermatozoa is unclear (The presence of viruses in semen is probably more widespread than currently appreciated, and the absence of virus in genital secretions should not be assumed for traditionally non–sexually transmitted viruses. The investigation of virus detection and persistence in semen across a range of viruses is useful for clinical and public health reasons, in particular for viruses that lead to high mortality or morbidity rates or to epidemics.Results of literature search for viruses that are capable of causing viremia and found in human semen.


We report the whole-genome sequence of the foot-and-mouth disease virus (FMDV) O/MOG/BU/2-7/2015 isolated in Mongolia in 2015. This virus is closely related to isolates identified in Southeast Asia in 2015 and is classified under the O/ME-SA/Ind-2001d lineage. This is the first detection of an FMDV of this lineage in Mongolia. Aphthovirus genus within the Picornaviridae family. Its genome is composed of a single strand of positive-sense RNA of approximately 8.4 kb in length, which is divided into S and L fragments by a poly(C) sequence at the 5′ untranslated region (UTR). FMDV is categorized into seven immunologically distinct serotypes , each of which can be divided into a variety of genetically distinct topotypes and lineages (Foot-and-mouth disease virus (FMDV) is classified under the The FMDV serotype O topotype Middle East-South Asia lineage Ind-2001 (O/ME-SA/Ind-2001) was detected and first defined in 2001 and has been the dominant lineage in the Indian subcontinent since 2008 . FMDV of4–In the present study, epithelial samples were collected from cattle that showed clinical symptoms of FMDV in Bayan-Ulgii province in Mongolia in March 2015. The FMDV O/MOG/BU/2-7/2015 was isolated from samples according to the World Organisation for Animal Health (OIE) manual . Viral RThe L-fragment sequence of O/MOG/BU/2-7/2015 was 7,754 nucleotides (nt) in length, and included a 5′ UTR of 685 nt, a single open reading frame (ORF) of 6,999 nt, and a 3′ UTR of 70 nt. The ORF was predicted to encode a polyprotein of 2,333 amino acids composed of four structural proteins, VP1 to VP4, and eight nonstructural proteins, L, 2A to 2C, and 3A to 3D. This virus isolate is the most closely related to O/BAN/GO/Ka-236(Pig)/2015 (GenBank accession no. KX712091) in the public database, with a 98.6% nt identity rate and no indels.In conclusion, the FMDV O/ME-SA/Ind-2001d was isolated from epithelial samples following an outbreak in 2015 in the western part of Mongolia. Our findings confirm the first incursion of FMDV O/ME-SA/Ind-2001d into East Asia. FMDV O/ME-SA/Ind-2001d has continued to spread into the Eurasian and African continents, indicating the need for epidemiological information and monitoring of this lineage to develop appropriate control strategies against this disease.LC320038.The genome nucleotide sequence of O/MOG/BU/2-7/2015 has been deposited in GenBank under accession no.


Picornavirales members from six different spider species found in Washington state. These six viral sequences distinctly clustered together phylogenetically with less than 35% amino acid identity to the closest reference viral genome.We report draft genome sequences of six novel  As new arthropod-borne zoonoses become of increasing relevance to human health it has become clear that greater understanding of the arthropod virome is required . Spider Hexura picea Simon is a native mygalomorph spider of the family Mecicobothriidae; Metellina curtisi (McCook) is a native orbweaver of the family Tetragnathidae; Hyptiotes gertschi Chamberlin & Ivie is a native triangle-weaving orbweaver. Theridion simile C. L. Koch is a native cobweb weaver of Holarctic distribution. Theridion tinctum  is an introduced cobweb weaver and Xysticus cristatus (Clerck) is an introduced crab spider, both of European origin.All spiders were sampled in western Washington and stored in ethanol for <1 year before sequencing. Metagenomic libraries were performed as described previously . LysatesTheridion simile spider, an 8,825 nucleotide (nt) contig with one continuous open reading frame was assembled corresponding to the novel Picornavirales Rainier virus UW1. The translated open reading frame aligned 29% by amino acid to Solenopsis invicta virus-1 (NC_006559)  . From thHexura picea spiders, two contigs of 9,798 and 10,025 nt were assembled with ORFs of 9,734 and 9,922 nt, respectively. The translated ORFs aligned 82% by amino acid, constituting two strains (BG1 and RC1) of the novel Burke-Gilman virus. They aligned 33% by amino acid to the polyprotein of the Rice tungro spherical virus, a member of the Secoviridae family  and aligned 35% by amino acid to the helicase of Aichi virus 1 .Partial genome sequences from three novel species from this likely novel clade were also recovered. Metagenomic sequences from a  virus 1 . The ThePicornavirales order as they cluster closer to each other than to any of the reference viral sequences listed here. Further taxonomic, sequencing, and functional studies would be required to fully delineate their place within the Picornavirales order.These viruses likely form a new clade within the KX779450 (Crab spider picornavirus J1), KX782311 and KX782312 (Burke-Gilman virus RC1 and BG1), KX782313 (Rainier virus UW1), KY316057 (Noveria virus B8-Tt), KY316058 (Volivirus B7-MC0), and KY316059 (Swagivirus HG B7-Hg).The sequences described in this study are deposited in DDBJ/ENA/Genbank under the accession numbers


Measurement of lumbar spine range of motion (ROM) is often considered to be an essential component of lumbar spine physiotherapy and orthopedic assessment. The measurement can be carried out through various instruments such as inclinometers, goniometers, and etc. Recent smartphones have been equipped with accelerometers and magnetometers, which, through specific software applications (apps) can be used for inclinometric functions.® app (TiltMeter© -advanced level and inclinometer) for measuring standing lumbar spine flexion–extension ROM in asymptomatic subjects.The main purpose was to investigate the reliability and validity of an iPhoneA cross-sectional study was carried out.This study was conducted in a physiotherapy clinic located at School of Rehabilitation Sciences, Iran University of Medical Science and Health Services, Tehran, Iran.A convenience sample of 30 asymptomatic adults  was recruited between August 2015 and December 2015.12–L1 and S1–S2 spinal levels. From this position, they were asked to perform maximum lumbar flexion followed by maximum lumbar extension with their knees straight. Two blinded raters each used an inclinometer and the iPhone ® app to measure lumbar spine flexion–extension ROM. A third rater read the measured angles. To calculate total lumbar spine flexion–extension ROM, the measurement from S1–S2 was subtracted from T12–L1. The second (2 hours later) and third (48 hours later) sessions were carried out in the same manner as the first session. All of the measurements were conducted 3 times and the mean value of 3 repetitions for each measurement was used for analysis. Intraclass correlation coefficient (ICC) models  and  were used to determine the intra-rater and inter-rater reliability, respectively. The Pearson correlation coefficients were used to establish concurrent validity of the iPhone® app. Furthermore, minimum detectable change at the 95% confidence level (MDC95) was computed as 1.96 × standard error of measurement × Following a 2–minute warm-up, the subjects were asked to stand in a relaxed position and their skin was marked at the T® app with ICC values of ≥0.85 and ≥0.85, respectively. The MDC95 ranged from 5.82°to 8.18°for the intra-rater analysis and from 7.38°to 8.66° for the inter-rater analysis. The concurrent validity for flexion and extension between the 2 instruments was 0.85 and 0.91, respectively.Good to excellent intra-rater and inter-rater reliability were demonstrated for both the gravity-based inclinometer with ICC values of ≥0.84 and ≥0.77 and the iPhone®app possesses good to excellent intra-rater and inter-rater reliability and concurrent validity. It seems that the iPhone® app can be used for the measurement of lumbar spine flexion–extension ROM.The iPhoneIIb. Measurement of lumbar spine range of motion (ROM) is routinely used by clinicians in order to assess patients with low back pain . Therefor ≥ 0.75) (r = 0.88), compared to extension (r = 0.42). r = 0.87), extension (r = 0.92), and lateral flexion (r ≥ 0.94) ROM (r = 0.76). Good reliability and validity of the inclinometer technique for measuring lumbar spine ROM have also been reported in other studies (An inclinometer or tilt meter is an instrument for measuring angles of slope (or tilt), elevation or depression of an object or body segment with respect to gravity . This in ≥ 0.75) . In addi.94) ROM . In anot.94) ROM . Newton  studies . Conside® and those that use the Android™) has brought a wide range of clinical measurement applications (apps) within the reach of most clinicians. The vast majority of smartphones have numerous built-in sensors such as accelerometers, magnetometers, and gyroscopes that make the phone capable of detecting joint positions and measuring joint ROM  which help clinicians measure joint ROM more accurately, easily, and quickly.The number of smartphone users is rapidly increasing worldwide, and it has been estimated that this number will rise to 2.5 billion people by 2017 . In addioint ROM . A numbe® app (iHandy© level) for measuring standing lumbar spine lordosis in 30 asymptomatic subjects. Good inter-rater and intra-rater reliability was reported for the inclinometer  and iPhone® app . The concurrent validity between the 2 instruments was good with a Pearson correlation coefficient (r) of 0.86 (® app (iHandy© level) in 30 asymptomatic subjects. In general, the results of the study indicate a good intra-rater and inter-rater reliability bubble inclinometry (ICC ≥ 0.81) and the iPhone® (ICC ≥ 0.80). The concurrent validity between bubble inclinometry and the iPhone® app was good with the ICC values of ≥ 0.86. However, within-day intra-rater reliability was not examined (® app (TiltMeter© -advanced level and inclinometer) for measuring standing lumbar spine flexion and extension ROM in asymptomatic subjects. The hypotheses of the study were as follows:To date, several studies have investigated the reliability and concurrent validity of these apps for measuring lumbar spine posture and ROM.  of 0.86 . In anotexamined . Therefo(a) There is no statistically significant difference between the reliability of the 2 instruments for measuring standing lumbar spine flexion and extension ROM.® app (TiltMeter© -advanced level and inclinometer) is a valid and reliable instrument to measure lumbar spine flexion and extension ROM.(b) The iPhonen = 15, females n = 15) met inclusion criteria and participated in the current study. All of the subjects were identified and recruited by posters, emails and word of mouth from the University and the surrounding community. The inclusion criteria were: (i) no history of low back pain during the last 6 months, (ii) age ≥ 18 years . Approval for the study was obtained from the Ethics Committee at the University of Social Welfare and Rehabilitation Sciences  (Tehran-Iran). The sample size used in the study was determined based on previous similar studies . Salamh 18 years ; (iii) a18 years ; (iv) no18 years ; (vi) no® model 5  with TiltMeter© -advanced level and inclinometer app  were used in this study. The TiltMeter© -advanced level and inclinometer app   and iPhoeter app  is a proFollowing the recruitment process, the subjects were brought into a physiotherapy clinic at the School of Rehabilitation Sciences  where they performed a warm-up exercise supervised by 3 raters  who were Ph.D. candidates of physiotherapy. The raters had more than 6 years of experience in physiotherapy clinical practice. The warm-up exercise needed approximately 2 min to complete and consisted of lumbo-pelvic rotations in a crook lying position (lower trunk rotation). The physiotherapist asked the subjects to rhythmically rotate the legs about 45° to the right and left. The subjects were also requested to keep their legs together and maintain the soles of the feet on the treatment table while doing the exercise  Kolber . In addi3 spinous process or L3–L4 spinal level  line was also used to find the S2 spinous process  in maximum lumbar spine flexion and extension position. Randomization in measurements was not used because the aim of the study was to investigate reproducibility, which requires a consistent physiological status (12–L1 level and then on the S1–S2 level at the extremes of lumbar spine flexion and extension (1–S2 was subtracted from the T12–L1. This technique of measurement was repeated with the iPhone® using the TiltMeter© -advanced level and inclinometer app. The raters (MRP and EJ) were blinded in this technique as well and the third rater (MT) read the measured angle from the TiltMeter© -advanced level and inclinometer app. After an interval of 2 hours, the procedure was repeated in the same manner.Following the warm-up exercise, the subjects were asked to stand in a relaxed position with their feet apart at shoulder width and their arms hanging loosely at their side. The intercrestal  or interal level . Once th process . Once theyeliner . The inceyeliner . The iPhtom side . The iPhl status  In neutrxtension  and 6. TThe third session (48 hours later) was started with a 2-minute warm-up exercise (same as the first session), and then the measurements were repeated again. In order to control confounding variables and to improve consistency among the placement of the instruments, one rater (MRP) was responsible for marking the subjects in all 3 sessions.All the measurements were conducted 3 times by the 2 raters and the mean value of 3 repetitions from each measurement was used for analysis.P < 0.05. Descriptive data are expressed as mean measurement angles ± standard deviations (SD) calculated for each series of measurements (® app) can be utilized with reliability and confidence among equally experienced clinicians . Prior to the statistical analyses, the Kolmogorov–Smirnov (K–S) test was performed to evaluate the normality of data distribution. For all tests, the statistical significance level was set at urements . The relinicians . Using S95) was computed as 1.96 × SEM The scale from nfidence . Furthernt error .® app was evaluated by using the Pearson correlation coefficient (r). The correlation coefficient can vary from −1 to +1. A value of 0 indicates that there is no linear relationship between the two continuous variables. A value greater than 0 indicates a positive relationship; that is, as one variable increases, the other variable also increases (direct relationship). A value less than 0 indicates a negative relationship; that is, as one variable increases, the other variable decreases (inverse relationship)  . To calcThe 95% limits of agreement were also calculated as mean difference ± 1.96 × SD . After tAmong the 49 subjects originally recruited, 19 were excluded based on the eligibility criteria: 16 had low back pain; 1 had a surgical instrument in the spine; and 2 had obvious scoliosis. Thus, 30 subjects were included in the current investigation.95 are reported in ® for lumbar spine flexion and extension ROM ranging from 0.84–0.92   .® values. However, individual differences may range from the iPhone® being 6.26° greater (the upper limit of agreement) to 6.86° less (the lower limit of agreement) than the gravity-based inclinometer for lumbar flexion ROM. In lumbar extension ROM, the individual differences may range from the iPhone® being 4.87° greater (the upper limit of agreement) to 5.39° less (the lower limit of agreement) than the gravity-based inclinometer.The 95% limits of agreement  and iPhone® app (TiltMeter© -advanced level and inclinometer) for measuring standing lumbar spine flexion and extension ROM in 30 asymptomatic subjects. The novelty of this investigation was that the within-day intra-rater reliability was evaluated as well. In addition, a new iPhone® app was utilized to measure isolated lumbar spine flexion and extension ROM.The aim of the current study was to evaluate the intra-rater and inter-rater reliability, MDC® app in less-experienced raters.Various disorders of the lumbar spine can affect the ROM, including ankylosing spondylitis, back strains, osteoarthritis, scoliosis, fractures, and spondylolisthesis . Therefo® apps . ® model 4 (iHandy© level app) and a bubble inclinometer in 30 asymptomatic subjects. The mean flexion measured by iPhone® for 2 raters was 49.5°, and in our study, the mean flexion measured by both raters was 51.5°. However, there is a small difference (∼6.6°) between the mean flexion measured using inclinometers reported by There are few published studies measuring the reliability and validity of iPhone® apps . In genetruments  and slig©-advanced level and inclinometer app with r values ≥0.85. The results of the validity are consistent with r values (≥0.86). However, the iPhone® app used in the previous studies was different from this study. The findings of the investigation confirmed the hypotheses that the TiltMeter© -advanced level and inclinometer app is a valid and reliable instrument for measuring standing lumbar spine flexion and extension ROM. Another point worth mentioning is that the external validity of the results could be limited since the rater A (MRP) was only responsible for marking the subjects.Validity refers to how well a measurement instrument actually measures the underlying outcome of interest . Accordi® app over the gravity-based inclinometer is that the app is available anytime, anywhere and can be downloaded for free from Apple’s App Store. Moreover, this app is also available for free for Android smartphones on AppCrawlr (http://appcrawlr.com/). However, it is important to consider limitations related to smartphone use. Clinicians may be reluctant to use their personal smartphone for assessment, because there would be a direct contact between the patient’s skin and the smartphone  was responsible for marking the subjects; therefore, the external validity could be limited.The subjects of the current study were asymptomatic with approximately normal body mass index (BMI). Thereby, the generalizability of these findings is limited and the data obtained from the asymptomatic subjects is not representative of the population with low back pain. Further research is needed to evaluate symptomatic subjects or individuals with different body morphology, because excess adipose tissue may affect the ability to accurately detect bony landmarks for instrument placement. In addition, only flexion–extension ROM was evaluated and not lateral flexion or axial rotation. Because in each of the 3 sessions the subjects were measured 3 times by the 2 raters at the TLast but not least, future research is encouraged to assess the reliability and validity of this app for measuring the ROM of other joints as well. Such an analysis would give insight on the application of smartphone devices in physiotherapy and orthopedic assessment.© -advanced level and inclinometer app is an inclinometer app of iPhone®. This investigation demonstrated that the app possesses good to excellent reliability (ICC ≥ 0.77) and concurrent validity with a gravity-based inclinometer (r ≥ 0.86) for measuring standing isolated lumbar flexion and extension ROM. According to the findings of the study, it seems that the app can be used for the measurement of lumbar spine flexion and extension ROM.Measurement of joint ROM is a part of physiotherapy and orthopedic assessment of various pathologies. Hence, numerous instruments have been introduced for this purpose . Smartphone inclinometer apps have been developed in recent years which allow clinicians to evaluate joint ROM more quickly and easily. The TiltMeter10.7717/peerj.2355/supp-1Data S1Click here for additional data file.


We report the results of pathologic examinations of 2 fetuses from women in Colombia with Zika virus infection during pregnancy that revealed severe central nervous system defects and potential associated abnormalities of the eye, spleen, and placenta. Amniotic fluid and tissues from multiple fetal organs tested positive for Zika virus. In October 2015, Colombia confirmed its first case of autochthonous Zika virus infection  with brain tissue loss, including absence of cerebral hemispheres . Amniotic fluid sampled at 17 weeks was positive for Zika virus by real-time reverse transcription PCR (rRT-PCR). The fetus was at 20 weeks of gestation when evaluated.Case 2 involved a 15-year-old pregnant resident of Bogotá who reported symptoms compatible with Zika virus disease, including generalized rash for 2 days and myalgia, after travel to a municipality with prevalent Zika virus infections, with onset occurring at 16–20 weeks of gestation. She had an uncomplicated obstetric history with 1 prior live birth. Prenatal care started in week 26; an ultrasound at that time showed a fetus with abnormal clefts in the cerebral hemispheres of the brain , with cleft walls separated and filled with cerebrospinal fluid (open lip). Follow-up ultrasound indicated large regions of parietal and temporal brain parenchymal loss. The fetus was evaluated and Zika virus diagnosis confirmed at 27 weeks of gestation.Autopsies of fetal organs and placenta with anatomic pathology and microscopic evaluation were performed for both fetuses. Tissue samples were fixed in 10% neutral-buffered formalin and fixed in paraffin; 4-μm-thick cuts were stained with hematoxylin and eosin for morphologic analysis with light microscopy. RNA was extracted from fresh tissue by using a TRIzol plus RNA purification kit . We used rRT-PCR for the detection of Zika virus (NS5) and 1-step RT-PCR for the detection of the envelope protein–coding region (360 bp) were performed as previously described , which highlights the potentially long window of vulnerability to adverse outcomes among pregnant women with Zika virus infection.–Persistent viremia in maternal serum has been previously reported (Given the epidemic levels of Zika virus disease being reported in Colombia and the severe outcomes associated with congenital infection, we call attention to the importance of healthcare providers reporting cases of Zika virus disease for adequate public health surveillance. Recommendations might include enhanced surveillance of acute cases among pregnant women and stillbirths and abortions related to Zika virus disease during pregnancy, as well as adverse outcomes among live births. Collecting information on congenital Zika virus infection is important to helping public health authorities effectively address this new health threat and make evidence-based decisions, which will benefit Colombia and other countries throughout the world.


Virus distribution and the proportion of organic material in horizontal transport versus processes in retention zones remain to be determined in detail. The need for up‐scaling the contribution of virus‐related processes in fluvial systems is of global relevance. Further, the role of climate change and the effect of anthropogenic alterations of fluvial systems on viruses require attention. The identification of these considerable knowledge gaps should foster future research efforts.The ecology of viruses has been studied only in a limited number of rivers and streams. In light of a recent re‐appraisal of the global fluvial surface area, issues such as abundance and production, host mortality and the influence of suspended particles and biofilms are addressed. Viral life cycles, potential impacts of viruses on water biochemistry and carbon flow, and viral diversity are considered. Variability in trophic levels along with the heterogeneous nature and hydrological dynamics of fluvial environments suggest a prevailingly physical control of virus‐related processes under lotic conditions and more biological control under lentic conditions. Viral lysis likely contributes to a pool of rapidly cycling carbon in environments typically characterized by high proportions of recalcitrant terrestrial carbon. On average, 33.6% (equalling 0.605 Pg C year I.et al., et al., et al., 2 outgassing was underestimated in the past  has been documented in the alkaline, hypersaline Mono Lake, California . Viruses are also involved in shaping the composition of bacterial communities, either by reducing abundant host taxa or by introducing new genetic information into their hosts. They may be the largest genetic reservoir on earth and may boost the resilience of ecosystems by sustaining multiple species with similar or identical biochemical pathways  an overview of existing information on virus ecology in fluvial waters by tackling issues such as virus‐mediated microbial host mortality, viral lifestyles and mechanisms controlling viral activity; (ii) an assessment of the impact of variable hydrology and the role of suspended particles and biofilms on viruses; (iii) information on the need to investigate horizontal transport processes of virus‐related solutes and particles, and virus dissemination; and (iv) arguments for the necessity of up‐scaling the contribution of virus‐related processes, for example their potential contribution to global CO2 outgassing and the role of climate change on these contributions. These considerations include the significance of anthropogenic alterations of fluvial systems and, together, will help define research fields for future studies.Surprisingly, for running water systems, many aspects of aquatic virus ecology are underrepresented or ignored. Recently, however, interest among limnologists and microbial ecologists is increasing  differences between marine, lacustrine and running waters affect the abundance, composition, production and control mechanisms of viruses. Generally, as a result of the balance between production and decay, virus numbers are linked to system productivity. Eutrophic water bodies typically contain more virus particles than oligotrophic systems  activity supports high viral production. In running water systems, information on viral production rates is conspicuously scarce, but the rates seem to vary over several orders of magnitude Table . ExampleIII.et al., et al., As in the marine environment, in inland aquatic systems viral infection of both auto‐ and heterotrophic bacteria and subsequent lysis of host cells is responsible for a considerable proportion of prokaryotic mortality  were unexpectedly extracted from human oropharyngeal samples; this virus caused harm to the human host  was found on suspended aggregates in a semi‐enclosed bay in the southwest lagoon of New Caledonia influenced by terrestrial input  in deeper layers of the benthos  is evident and should be addressed in future work.V.via protozoan grazing, most of the carbon will be channelled to higher trophic levels . By contrast, if viral lysis accounts for most prokaryotic losses, then carbon and nutrients are diverted away from larger organisms  was variable, averaging 7.1%; in reservoirs fed by the Senegal River the percentage was much lower  flushed section (25.0%) and in the main channel (21.2%)  pool at the expense of bacterio‐ and phytoplankton grazing  may pass the viral loop. Combining this with a global appraisal of CO2 evasion data for streams and rivers  to the globally respired carbon. For comparison, in the oceanic environment the viral shunt was estimated to release 0.37–0.63 Pg C year−1 on a global scale . Even in cases where viral lysis releases only a small fraction to the large fluvial organic carbon pool, it could still contribute significantly to the readily utilizable carbon (labile and semi‐labile fraction). This is particularly relevant in these systems, which are typically characterized by a high proportion of allochthonous  aged and recalcitrant carbon. In Danube floodplain areas, hydrological events substantially influence dissolved organic matter (DOM) composition in backwaters, and DOM quality is an important factor regulating bacterial production . This often leads to a continuum of trophic levels, from oligo‐ to eutrophic subsystems on narrow spatial and temporal scales  DOC through the viral lysis of infected hosts. This involves promoting the uptake and respiration of material released from lysis by non‐infected cells. The magnitude of re‐mineralization through a bacteria–virus loop depends strongly on the number of passes through the loop, with increasingly more DOC respired at each turn  water surface area and a gas transfer velocity. A recent estimate of the global CO2 emission from streams and rivers predicts about 70% of the flux occurring over just 20% of the land surface  One important driving factor in fluvial systems is their highly dynamic and heterogeneous nature, compared to the marine or lacustrine realm. Variable hydrology, edaphic heterogeneity and the broad range of trophic levels  are essential elements of running waters, apparently impacting virus ecology significantly.(2) Suspended particles, aggregates and biofilms are typical features of flowing waters; they probably influence virus‐related processes more strongly than in most lacustrine and oceanic systems. These structures can affect microbial  diversity, food‐web structure and biogeochemical cycles.2 outgassing from fluvial systems, synthesized from the scattered literature, reveals a potentially significant contribution on a global scale.(3) Through repetitive viral lysis of infected bacterial hosts, the bacterial–viral loop appears to be significantly involved in removing part of the recalcitrant, terrestrial, dissolved organic matter from the water; increasingly more material is respired at each turn of the loop. A first appraisal of the viral contribution to CO(4) Horizontal transport processes are relevant for virus dissemination and for virus‐influenced solutes and particles. Targeted research on such horizontal processes  is an important goal for the future.(5) The significance of extensive anthropogenic alterations of fluvial systems, and of disturbance events by flooding, to viral lifestyles, host–virus diversity and virus activity needs to be investigated in detail, also in light of the effects of climate change.(6) We need to develop comprehensive research programmes for a better understanding of virus‐related processes in flowing water systems. The outcome of such studies can be expected to contribute significantly to modern concepts of fluvial systems. Such concepts are necessary to understand and manage this important water resource better.


TB) cases and 1.4 million deaths worldwide. Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the vaccine available against TB, but it is insufficient for global TB control. This study evaluated the immunogenicity of the Mycobacterium tuberculosis antigen Rv1626 in mice while assessing the effect of co‐delivering either Cpe30 (immunostimulatory peptide), CS.T3378–395 (promiscuous T helper epitope) or flagellin (TLR5 agonist) or a combination of all three immunostimulatory agents. Rv1626 and the respective immunostimulatory proteins/peptides were co‐displayed on polyhydroxybutyrate beads assembled inside an engineered endotoxin‐free mutant of Escherichia coli. Mice vaccinated with these beads produced immune responses biased towards Th1‐/Th17‐type responses, but inclusion of Cpe30, CS.T3378–395 and flagellin did not enhance immunogenicity of the Rv1626 protein. This was confirmed in a M. bovis challenge experiment in mice, where Rv1626 beads reduced bacterial cell counts in the lungs by 0.48 log10 compared with the adjuvant alone control group. Co‐delivery of immunostimulatory peptides did not further enhance protective immunity.In 2015, there were an estimated 10.4 million new tuberculosis ( The synthesis of the most common one, polyhydroxybutyrate (PHB), a short chain length PHA, involves three key enzymes, β‐ketothiolase (PhaA), acetoacetyl‐CoA reductase (PhaB) and polyester synthase (PhaC). The knowledge about topology, structure and properties of PhaC, which remains covalently linked to the PHB granules, has allowed translational fusion of foreign proteins to produce functionalized beads. These beads have been used for biomedical applications like protein production and purification as well as diagnostics and vaccines . Such a vaccine needs to be affordable and accessible for developing countries, safe and able to elicit a longer lasting immunity than the current vaccine, Bacille Calmette–Guérin  displayed on beads and formulated in DDA (dimethyldioctadecylammonium). IgG1 and IgG2c responses in sera were analysed by ELISA Fig. . IgG1 reet al. , where h et al., . HoweverSerum IgG1 and IgG2c responses to Rv1626 in mice vaccinated with Rv1626 beads with or without addition of immunomodulators were evaluated by ELISA Fig. . These rM. tuberculosis or M. bovis infection, the ability of Rv1626 displayed on beads and combined with immunomodulators Cpe30 or CS.T3 to protect against tuberculosis was evaluated in a M. bovis challenge experiment. These beads were selected from the immunogenicity experiment as they induced higher cytokines levels than the other vaccines.As there are no confirmed correlates of protection for Additional groups were given soluble recombinant Rv1626 or BCG while a control group received DDA. Antibody responses of mice vaccinated with the selected PHA bead‐based vaccines are shown in Fig. et al. The induced IgG subclasses provide an indication of the contribution of Th1‐ or Th2‐type cytokines in the response; thus, release of IgG1 is related to induction by Th2‐type cytokines, whereas production of IgG2c is related to induction by Th1‐type cytokines (Petrushina −1 of soluble recombinant Rv1626 or bovine PPD (purified protein derivative), and results are shown in Fig. Splenocytes from mice were stimulated with 5 μg ml10 and 0.98 log10, respectively, compared to the negative control (DDA). Vaccination with the Rv1626 beads reduced the lung bacterial counts by 0.48 log10. Although this reduction was not significant compared to the negative control, in this group, lung bacterial counts were not significantly different compared to those for the BCG‐vaccinated group. Derrick et al.  vaccinated subcutaneously three times at 9 day intervals with different doses of Rv1626 displayed on beads and Wt beads.Fig. S2. Serum IgG1 and IgG2c titres, expressed as EC50 values in mice (8 per group) vaccinated subcutaneously three times at 9 day intervals with Rv1626 beads or Rv1626 beads displaying different immune modulators.Fig. S3. Cytokine responses of mice splenocytes upon stimulation with soluble Rv1626 (recRv1626) and analysed by cytometry bead array.Click here for additional data file.


Viruses are genetically diverse, infect a wide range of tissues and host cells and follow unique processes for replicating themselves. All these processes were investigated and indexed in ViralZone knowledge base. To facilitate standardizing data, a simple ontology of viral life-cycle terms was developed to provide a common vocabulary for annotating data sets. New terminology was developed to address unique viral replication cycle processes, and existing terminology was modified and adapted. The virus life-cycle is classically described by schematic pictures. Using this ontology, it can be represented by a combination of successive terms: “entry”, “latency”, “transcription”, “replication” and “exit”. Each of these parts is broken down into discrete steps. For example Zika virus “entry” is broken down in successive steps: “Attachment”, “Apoptotic mimicry”, “Viral endocytosis/ macropinocytosis”, “Fusion with host endosomal membrane”, “Viral factory”. To demonstrate the utility of a standard ontology for virus biology, this work was completed by annotating virus data in the ViralZone, UniProtKB and Gene Ontology databases. What could be more alien than a virus? These parasitic entities evolve at the periphery of cellular organisms, and have developed unique methods to replicate and disseminate their genetic material. Many of these unique molecular processes may find their root in ancient biochemistry, down to the RNA world . Indeed In this work the SwissProt virus annotation team addressed the annotation and classification of all major means used by eukaryotic viruses to achieve their parasitic life-cycle. An extensive study of viral textbooks and the recent literature was performed to identify essential and conserved viral life-cycle steps. This study has focused on processes directly involved in entry, expression, replication and exit of the viral genetic material. Host-virus interactions implicated in immunity have been covered in previous publications ,3. DespiThe 82 terms describing the core viral replication cycle were used to annotate virus entries in ViralZone , UniProtViralZone is a database that links virus sequence with protein knowledge using human-readable text and controlled vocabularies . This weUniProtKB is a comprehensive resource for protein sequence and annotation data . All knohttp://www.ebi.ac.uk/GOA/Keyword2GO).A more complex and widely used vocabulary is that of the Gene Ontology (GO) in which relations between terms have a number of explicit meanings which can be used to make further inferences–such as eukaryotic transcription factors may be located in the nucleus ,7. GO anThe virus replication cycle core terms have already been implemented in these three resources by over 12,000 manual and 2,000,000 automatic annotations. This work provides a basal knowledge of virus protein function that can be used as a reference for similar sequences, thereby facilitating analysis of large scale datasets with viral protein expression.This work describes the creation of a virus life-cycle vocabulary in ViralZone, UniProtKB and Gene Ontology. Inter-relations between vocabulary and ontologies, and the way virus sequences are curated using this system have been described in a previous publication .The first step of this work was to identify all specific steps used by eukaryotic viruses during their life-cycle. To do so, an exhaustive review was performed in virology textbooks, published reviews, and existing ontologies by the UniProtKB/Swiss-Prot virus team. All the processes identified were structured into chronological steps involved in virus entry, transcription/replication/translation and exit. This led to the creation of 69 ViralZone pages describing most of the identified vocabulary . The VirThe GO team at the EBI collaborated with the UniProtKB/SwissProt team to update and complete the GO database with the virus life-cycle molecular processes. The mapping effort led to the update of 56 GO terms and the development of 14 new GO terms . 58 of tUniProtKB keywords summarize the content of a UniProtKB entry and facilitate the search for proteins of interest. Using ViralZone vocabulary we created 30 keywords (KW) and updated 11 KW  for a toTo demonstrate the utility of a standard ontology for virus biology, this work was completed by annotating virus data in the ViralZone, UniProtKB and Gene Ontology databases. Expert curation has been done in different ways. In UniProtKB/Swiss-Prot, keywords were manually introduced in viral entries after careful reading of the literature, using an editor available only to UniProtKB curators. All keywords with a related GO term  were autThis works follows the events describing the fate of viral genetic material during the three stages of the infectious cycle: entry, genome expression/replication, and exit.Virus entry starts with virion attachment to the host cell, leading to the uptake of the viral nucleic acid into a target cellular compartment in which it will start transcribing and replicating. The second step is transcription of viral genes, leading eventually to replication of the viral genome. Latency consists in a pause at the start of the transcription step; the viral genome is either silenced or transcribes few genes, putting on hold the resolution of the transcription/replication step. When this hold is released, the viral genome proceeds to the completion of this second step without going back to latency. The last step is virus assembly and exit. This corresponds to late transcription in most viral genomes. Often the virus will overproduce genomic and structural materials to assemble as many virions as possible. This can lead to irreversible damage to the host cell.In the following paragraph, viral processes discussed in the text are underlined when they correspond to a vocabulary or ontology term. The corresponding ViralZone pages can be retrieved by typing the start of the term in the ViralZone search box and choosing the right name.“Virus entry” refers to all the steps happening between the extracellular virion up to the transport of viral genetic material to the site of transcription/replication  8]. The. The8]. Viruses can infect new cells by many means. Some viruses exploit “cell to cell transport”. This includes plant plasmodesmata , nanotubThe most classical route of infection is through an external virion particle that has to cross the cellular membrane to deliver its genetic material into the cell. The very first step is “viral attachment to host cell”, by binding surface molecules such as glycans or proteins . AttachmEndocytosis is an event whereby virion interaction with an entry receptor triggers active uptake of the virion by the cell to be brought to endosomes. The virus exploits an existing endocytic pathway to gain access to cellular internal compartments in early endosomes, late endosomes or even lysosomes from where it will be able to inject its genetic material into the cytoplasm. The nature of the host entry receptor bound by a virion likely determines which of the many routes of endocytosis it will use. There are four major routes: “clathrin-mediated endocytosis”, “caveolin-mediated endocytosis”, “lipid-mediated endocytosis” and “macropinocytosis” . InteresThe endocytosed virion will then deliver its genetic material into the host cytoplasm often by exploiting the low pH endosomal environment. Enveloped virions will trigger “fusion of virus membrane with host endosomal membrane” , non-envThe viral genetic material delivered into the host cell cytoplasm is often addressed to a specific cellular location, either by “actin-dependent inwards transport” or “microtubule dependent inward transport” . This trAt the end of virus entry step, the virus genome can either start transcribing/replicating leading to the formation of new progeny, or it may enter a latency mode. This mode is characterized by very low transcription of latent genes. The virus can stay dormant in the host cell for years before being activated by an external event .Viral genome expression is the second step of the infectious cycle, which often precedes “viral replication”. The nature of the genome is the critical point that determines the mechanism of transcription and replication. Therefore we have represented the different genetic expression/replication processes using the Baltimore classification  22]. Th. Th22]. Viral dsDNA templated transcription is performed by classical cellular mechanisms, or the viral equivalent of it. To improve coding capacity, cellular splicing is exploited by dsDNA viruses that transcribe in the host nucleus. There are at least seven ways to replicate the genome of viruses having a dsDNA intermediate. The classical cellular “bi-directional replication”   can be rSs(+)RNA and dsRNA viral genomes are transcribed by viral RNA-dependent RNA polymerases from a dsRNA template. Interestingly, “ss(+)RNA replication” and transcription are similar, in that the same genomic mRNA is the template for translation and replication.Within eukaryotic cells, dsRNA is a strong inducer of antiviral-defense. Therefore RNA viruses hide their dsRNA template or prevent its formation: ss(+)RNA virus transcription/replication happens in membranous vesicles , whereasViroids and the hepatitis delta RNA genome consist of a partially double-stranded closed circular RNA molecule. Interestingly, “Viroids and hepatitis D replication” and “hepatitis D transcription” are assured by the host DNA dependent RNA polymerase, that is exceptionally able under these circumstances to use a RNA template .After replication/ transcription, viral mRNA is translated to produce viral proteins, but no known virus encodes for any translation machinery. Indeed, viruses can be defined as replicative genetic elements that do not encode ribosomes. The absence of a translation system is what defines their very parasitic nature. Therefore, viral translation is performed by host cellular machinery, and follows classical cellular mechanisms. Nonetheless, viruses trick host ribosomes in many ways to enhance the protein expression from their small genomes. This includes: “leaky scanning” , “ribosoAfter the replication phase, viruses express movement and/or structural proteins as means to export their genomes out of the cell . “Viral The easiest way for a virus to exit the host cells it to induce its death or lysis. This can occur naturally as for corneocytes (papillomaviruses) , or be iTo physically exit from the cell, the viral particle or genome have to be transported to the plasma membrane or to the cellular exocytosis machinery. Nuclear virus genomes migrate to the cytoplasm by “nuclear pore export”  , or buddThe first application of the viral ontology is to allow comprehensive annotation of virus genes and sequences in databases. Moreover, developing an ontology is akin to defining a set of data and their structure for other programs to use. Computers programs can use ontologies as data in any of their analysis. Therefore, the viral ontology gives computers access to a kind of expert knowledge analysis that can be essential in research. For example, Brandes et al. have recently used ViralZone capsid ontology data in their statistical analysis about gene overlapping and size constraints in the viral world . MoreoveThe virus replication cycle vocabulary and ontology have been expanded by collaboration between the Swiss-Prot and GO teams. These vocabulary and ontologies are all linked together and describe the mechanisms involved in eukaryotic viruses’ life-cycles. While most of our current knowledge is covered by these terms, our systematic approach will allow for expanding and updating the system. One achievement of this work is that it allows a virus’ life-cycle to be described by a succession of controlled vocabularies. This provides a means to store and manage knowledge in biological databases. For example, Zika virus life-cycle can be summarized by cutting this cycle into steps described by controlled vocabulary: “Attachment”, “Apoptotic mimicry”, “Viral endocytosis/ macropinocytosis”, “Fusion with host endosomal membrane”, “Viral factory”, “dsRNA-templated transcription/replication”, “Cytoplasmic capsid assembly”, “Viral budding via the host ESCRT complexes”, “Virus budding by cellular exocytosis”. These successions of terms describe accurately the pathway followed by the Zika virus genome across an infected cell. It uses ViralZone controlled vocabulary because some processes cannot be described by GO or UniProtKB ontologies when they cannot be associated with a gene. For example “Apoptotic mimicry” cannot be related to a viral gene or protein, as it involves the virion membrane.viralzone@isb-sib.ch) or Gene Ontology (http://geneontology.org/contributing-go-term). Several research institutes and public databases have initiated projects involving the annotation of viral genomes, and we hope that the terms and ontologies presented in this article, which are available from the ViralZone, UniProtKB and GO websites, will help them in these efforts.Our efforts to create a eukaryotic virus ontology have led to three levels of implementation: global knowledge and facts in ViralZone pages; viral protein annotation in UniProtKB through keywords; and viral gene and protein annotation through GO terms. This has led to the creation of 69 new ViralZone pages, at least 30 new SwissProt keywords and 59 new GO terms. At the time of writing (May 2016) the keywords provide a total of 2,348,548 annotations in UniProtKB while the equivalent GO terms provide 5,864,073 annotations. Together these three implementations provide a global view of viral biology, and a means to annotate knowledge, for a wide user community. Research groups may contribute to this viral ontology by providing suggestions for updating terms (e.g. requests for new terms) either through ViralZone (


Aedes aegypti and Ae. albopictus adult female mosquitoes; ≈1/84 larvae tested were Zika virus–positive; and rates varied among mosquito populations. Thus, vertical transmission may play a role in Zika virus spread and maintenance.To determine the potential role of vertical transmission in Zika virus expansion, we evaluated larval pools of perorally infected  Flaviviridae, Flavivirus) has continued to expand its distribution throughout the Pacific region and, since 2014, the Americas  and Ae. albopictus mosquitoes  to Zika virus through infectious blood meals and evaluated the mosquitoes’ capacity to transmit the virus to progeny. For this study, we used the Zika virus strain ZIKV HND , passaged once on C6/36 cells, and Zika virus PR , passaged 4 times on Vero cells and twice on C6/36 cells. Zika virus was propagated on C6/36 cells for 4 days, and freshly harvested supernatant was mixed 1:1 with sheep blood  and 2.5% sucrose.We exposed laboratory colonies of Infectious blood meals were offered to 4- to 7-day-old female mosquitoes, and weekly noninfectious blood meals were offered after the first oviposition. Eggs laid during the second oviposition and beyond were collected and hatched for subsequent testing. Third- to fourth-instar larvae were collected in pools of 5 and processed by homogenization and centrifugation. After RNA extraction, we used Zika virus–specific quantitative reverse transcription PCR  and establishes that Ae. albopictus mosquitoes are capable of vertical transmission of Zika virus in the laboratory.We tested 104 10 PFU/mL), resulting in >93% of disseminated infections and development of high viral titers in individual mosquitoes, averaging 7.1  to 7.5 (Ae. aegypti) log10 PFU/mosquito (Although the bypassing of the midgut during inoculation generally results in higher levels of vertical transmission, we fed mosquitoes high virus doses (8.9–9.3 logmosquito . Althougmosquito , suggestAe. aegypti and Ae. albopictus mosquitoes. Although the mechanism of vertical transmission with flaviviruses is generally thought to be infection of eggs during oviposition, rather than transovarial transmission (Together, these results indicate that Zika virus has a relatively high capacity for being transmitted vertically by both


The structure of long-term care (LTC) for old people has changed: care has been shifted from institutions to the community, and death is being postponed to increasingly old age. The aim of the study was to analyze how the use and costs of LTC in the last two years of life among old people changed between 2002 and 2013.N = 427,078). The costs were calculated using national unit cost information. Binary logistic regression and Cox proportional hazard models were used to study the association of year of death with use and costs of LTC.Data were derived from national registers. The study population contains all those who died at the age of 70 years or older in 2002–2013 in Finland (The proportion of those who used LTC and the sum of days in LTC in the last two years of life increased between 2002 and 2013. The mean number of days in institutional LTC decreased, while that for sheltered housing increased. The costs of LTC per user decreased.Use of LTC in the last two years of life increased, which was explained by the postponement of death to increasingly old age. Costs of LTC decreased as sheltered housing replaced institutional LTC. However, an accurate comparison of costs of different types of LTC is difficult, and the societal costs of sheltered housing are not well known. The use of long-term care (LTC) among old people is concentrated into the last years and months of life, and the use of LTC at the end of life is higher in older old than younger old age –4. DeathA number of political programs in Finland have attempted to change highly institutionalized LTC practices and have recommended community-based services instead , 7, as hThere are three types of round-the-clock LTC in Finland: inpatient care in health center wards , residential homes, and sheltered housing with 24-h assistance (referred to hereafter as sheltered housing). Of these, health centers and residential homes are institutional settings, and sheltered housing provides housing and closely related services. In the past, these LTC facilities were presented as forming a hierarchy: people with the smallest care needs were predominantly cared for in sheltered housing, and those with the greatest needs in health centers . HoweverMunicipalities are responsible for providing LTC for their citizens in Finland . The larWe describe and analyze the use and societal costs of round-the-clock LTC among old people during the last two years of life, and how those uses and costs changed between 2002 and 2013. These were analyzed for long-term care in total, and separately for health center inpatient wards, residential homes, and sheltered housing. During the study period the structure of LTC continued to change so that care was shifted from institutions to the community, and death was being postponed to increasingly old age. These changes are current in many countries, and the results of this study add knowledge about LTC use in a group with the highest care needs. This study was conducted as part of the project entitled “New Dynamics of Longevity and the Changing Needs for Services” (COCTEL).The study population was drawn from the Causes of Death Register (Statistics Finland). It consists of all those who died at the age of 70 years or over in 2002–2013 in Finland. The cutoff age of 70 was chosen because both the risk of death (Statistics Finland) and healthcare expenditures per resident start to increase at around 70 years of age in Finland . Use of The data on LTC use were derived from the Care Register for Healthcare and Care Register for Social Welfare . The information from these registers was linked using unique Personal Identification Codes. A more detailed description of the data collection has been given elsewhere . Days inPermission to access and use the register data was obtained from both register authorities. The research plan was approved by the Pirkanmaa Hospital District Ethics Committee.Use and costs of round-the-clock LTC were analyzed in total and separately for three types of LTC: (1) health center inpatient wards, if the person had a continuous length of stay of 90 days or over; (2) residential homes; (3) sheltered housing with 24-h assistance. The LTC in total is the sum of these three types of LTC.Three outcome variables were created for LTC in total and for each type of LTC: (1) any use, where 1 = used at least once in the last 730 days of life, and 0 = did not use in the last 730 days of life; (2) days in care ; (3) costs of care in the last 730 days of life.We multiplied the number of days in different types of LTC by their daily unit costs, derived from a national report  Table 11. The unThe unit cost of sheltered housing included staff, meals, and materials, but excluded housing, medicines, and some purchased services . InsteadAnalyses were performed for the whole study population and by age group . Chi-square tests were used to test the differences in the proportions of service users and genders between the years of death. One-way ANOVA was used to assess the change in the mean age at death during the study period. Independent samples median tests were used to test the differences in the number of days in care, and in the costs between those who died in different years. The number of days in and costs of LTC were analyzed among the users of services. In addition, the sum of days in LTC is presented. Binary logistic regression analyses were performed to assess the probability of using LTC by year of death. Age and gender were adjusted for.The costs of LTC in our data include a lot of zeroes, and the distributions are U-shaped, meaning that there were many individuals with few or no days in care  and many who were in care for 730 or almost 730 days. Survival methods such as Cox proportion hazard models may be employed when the data are skewed or multimodal, have heavy tails, or consist of excess zeroes . These mWe employed Cox proportional hazard models to analyze the development of the costs of LTC per user between 2002 and 2013. Costs were considered a “survival time” variable, and those who did not have “survival time”, i.e. whose costs were 0, were dropped from the models. None of the observations was treated as censored, as for all the end point was death. Age, gender, and year of death were independent variables. In addition, models including an interaction term (age * year of death) were run to find out whether the effect of age on costs differed between the years. The negative coefficient estimate (hazard ratio (HR) <1.00) for the proportional hazard model indicates a decreased hazard of reaching total costs, hence an increase in total cost . Therefop < .001), and the proportion of women decreased from 59.1% to 56.5% (p < .001). The number of people who died at the age of 70–79 years decreased and that of older people increased during the study period  over the study period among those who died at the age of 70+. It increased in younger age groups, but decreased (p < .001) among the oldest (90+) , while the use of sheltered housing increased (p < .001) in all age groups.The proportion of those who used LTC in total increased (0+) Fig. . The useThe likelihood of using LTC in total remained approximately at the same level between 2002 and 2013 when age was adjusted for   in the study period, but the costs per user decreased (p < .001)  from 2002 to 2013. Conversely, the number of days in and costs of sheltered housing increased (p < .001).Among those who used LTC, the number of days in LTC in total increased slightly  to find out whether the effect of age on LTC costs changed during the study period . The effect of this interaction term was statistically significantly associated with the costs of LTC in total for the years 2010–2013  0.993, 0.999), and with the costs of residential homes in 2012 and 2013 . This indicates that in these years the effect of age on LTC costs was 0.4% and on residential home costs 0.7% weaker than in 2002.The aim of this study was to describe and analyze how the use and costs of round-the-clock LTC among old people in the last two years of life changed between 2002 and 2013. We found that the proportion of LTC users increased among those who died at the age of 70 years or over, but the increase was not clear when age was adjusted for. This implies that the increase was due to the change in the age structure of old people, i.e. the postponement of death to older ages. Use and costs of LTC are higher among older old than younger old people, and the effect of age on use and costs of LTC did not change much during the study period. As the last years of life are being lived at a greater age than before, functional and cognitive disability is probably greater than previously , and theThe composition of LTC changed during the study period. The use of institutional LTC  decreased, and the use of sheltered housing increased. The number of days in LTC in total among users did not change much, but the costs of LTC in total per user decreased. This was mainly due to the replacement of institutional LTC with sheltered housing, the costs of which are lower than those of institutional LTC. However, the unit costs of sheltered housing are difficult to estimate, as the costs and contents of services vary between sheltered housing units. In particular, the costs that clients pay out of their own pockets are not well known, and not all of them are included in the unit costs used here. We used the national unit costs, but since these do not include the costs of medicines and housing, we estimated their impact on costs. Consequently, the difference between the daily costs of residential homes and sheltered housing diminished from €57 to €39. However, the unit cost of sheltered housing is probably an underestimation.The costs of LTC naturally depend heavily on the unit costs used. We used the same unit costs (2013) for all years, although costs may have changed during the study period. Furthermore, if there were significant changes in the content of care, or in the need for care among users, this may also have led costs to change. The unit costs used here are the national average costs, while the actual costs vary somewhat between municipalities and service providers .The role of sheltered housing clearly increased during the study period in the care of old people in their last two years of life, as in the care of old people in general –12, 30. According to our previous analyses, old people are not living until their deaths in sheltered housing as often as in residential homes. Also, the number of transitions to different care facilities  in the last year of life has been higher from sheltered housing than from residential homes , 32. In The use of registers, which are considered reliable , 35, is The focus of this study was on round-the-clock LTC; thus home care and informal care, which are important parts of LTC, were not included. The proportion of home care users in the last two years of life increased from 19% in 2002 to 21% in 2008 in Finland . AlthougIndividual characteristics, other than age and gender, were not controlled for in our analyses, although disease and disability , 39, livA remarkable change in the arrangement of round-the-clock LTC for old people in the last two years of life took place in Finland between 2002 and 2013. The number of days in LTC increased slightly, but the costs of LTC in total per user decreased during the study period. There was a shift from institutional care to sheltered housing. However, reliably comparing the costs of different types of LTC is difficult, because they do not include the same cost items. We estimated the societal costs of sheltered housing, but still could not include them all. In particular, the costs paid by the service users are difficult to estimate. The increased use of sheltered housing raises concerns about shifting the financial responsibility onto clients. Use of LTC in total increased, as the age structure of the decedents changed. As death continues to be postponed to older ages, the need for, and the use and costs of, LTC will probably increase. Choices should be made about how to supply good care for old people in ways that will share the costs of LTC equitably.


Lancet Series commissions. As use of the LiST model increases, many have expressed a desire to cost interventions within the model, in order to support budgeting and prioritization of interventions by countries. A limited LiST costing module was introduced several years ago, but with gaps in cost types. Updates to inputs have now been added to make the module fully functional for a range of uses.Achieving the Sustainable Development Goals will require careful allocation of resources in order to achieve the highest impact. The Lives Saved Tool (LiST) has been used widely to calculate the impact of maternal, neonatal and child health (MNCH) interventions for program planning and multi-country estimation in several This paper builds on previous work that developed an initial version of the LiST costing module to provide costs for MNCH interventions using an ingredients-based costing approach. Here, we update in 2016 the previous econometric estimates from 2013 with newly-available data and also include above-facility level costs such as program management. The updated econometric estimates inform percentages of intervention-level costs for some direct costs and indirect costs. These estimates add to existing values for direct cost requirements for items such as drugs and supplies and required provider time which were already available in LiST Costing.Results generated by the LiST costing module include costs for each intervention, as well as disaggregated costs by intervention including drug and supply costs, labor costs, other recurrent costs, capital costs, and above-service delivery costs. These results can be combined with mortality estimates to support prioritization of interventions by countries.The LiST costing module provides an option for countries to identify resource requirements for scaling up a maternal, neonatal, and child health program, and to examine the financial impact of different resource allocation strategies. It can be a useful tool for countries as they seek to identify the best investments for scarce resources. The purpose of the LiST model is to provide a tool to make resource allocation decisions in a strategic planning process through prioritizing interventions based on resulting impact on maternal and child mortality and morbidity.The online version of this article (10.1186/s12889-017-4738-1) contains supplementary material, which is available to authorized users. Significant progress was made toward reaching the Millennium Development Goals (MDGs) for improved maternal, neonatal, and child health (MNCH), including reducing the number of child deaths globally from 12.7 million in 1990 to 6 million in 2015; reducing the number of underweight children from 28% of those under age 5 in 1990 to 14% in 2014; and reducing the number of maternal deaths from approximately 523,000 in 1990 to an estimated 303,000 in 2015 [in The Lancet Series on Childhood Pneumonia and Diarrhoea [The Lancet Series on Maternal and Child Nutrition [The Lives Saved Tool (LiST) has been used widely to calculate the impact of MNCH interventions –8, in Thiarrhoea  and in Tutrition . As use utrition . Here, wResults generated by the LiST costing module include total costs for each intervention, as well as disaggregated costs by intervention including drug and supply costs, labor costs, other recurrent costs, capital costs, and above-facility costs.The LiST costing module is intended for use at the policy and strategic planning level, focusing on resource allocation decisions within MNCH programs. It can also be used to substantiate investment cases for MNCH and to support concept note development for the Global Financing Facility. It provides an estimate of the resources needed to implement an intervention or a package of services, but does not provide activity based costing, costing of program activities, or health systems strengthening requirements.The purpose of LiST is to provide a tool for making resource allocation decisions in a strategic planning process by prioritizing interventions based on resulting impact on maternal and child mortality and morbidity; LiST Costing adds a cost dimension which provides a means to estimate resources required and the ability to compare costs for different packages of interventions. See Additional file Questions can be explored such as: How much funding is required to achieve the goals of the strategic plan? What goals can be achieved with the current resources? What is the impact of alternative patterns of resource allocation in terms of both the associated costs and achieved goals of the strategic plan?By using LiST Costing in conjunction with the standard LiST module, scenarios can be developed by varying parameters such as costs inputs, coverage rates of interventions, and/or other inputs, which can then be evaluated based on the impact on maternal and child mortality and morbidity and cost associated with delivering the package of services. Results from different scenarios can be displayed easily for comparison purposes. Applications can also be re-visited in subsequent years for evaluation purposes or can be updated to inform a new priority-setting exercise.Impact analysis that addresses costs can be used to strengthen a country’s investment case for the Global Financing Facility or other donors, as well as contribute to discussions around domestic resource mobilization and identification of health program investment priorities. The results can be used beyond planning for the MNCH sector, including feeding into the development of a national health sector strategy, or even beyond the health sector to position the health budget within the broader government budget.The objective for developing the LiST costing module was to provide a means for estimating the financial cost of providing a service, while ensuring as much consistency as possible with data already available in LiST, as well as consistency with other methodologies followed by the WHO, such as the OneHealth Tool. Thus an econometric analysis was performed to estimate the contribution of intervention cost components that were not available in the OneHealth Tool. In addition, further methodological development of the LiST costing module was undertaken, including incorporating above-facility level costs into the existing intervention-level costs.target population - is the population on which the health intervention is focused, such as pregnant women or children aged <1 month;population in need – refers to the percentage of the target population that requires the intervention, such as the percent of pregnant women who need management of pre-eclampsia. For diseases such as diarrhea or malaria where there may be more than one case per year; this can be reflected in a percentage greater than 100%;coverage – refers to the effective coverage, i.e. the percentage of the population in need that actually receives the servicetreatment inputs – refers to drugs and supplies, medical personnel time requirements, and number of outpatient visits and inpatient days per case. Treatment inputs can be varied by delivery channel or level of service delivery, in order to reflect variation in drugs and supplies, skilled personnel, and other items that might be required when an intervention is delivered at higher levels of a health system.costs per service – calculated based on treatment inputs, and the unit costs for drugs and supplies, provider time, and costs of inpatient days and outpatient visitsThe impact estimates of LiST use coverage, effectiveness values and affected fractions to calculate mortality reductions or nutritional status improvements . LiST coIn the LiST costing module, the final cost per service is calculated as the multiplication of all of these factors :Number of services = Target population * % population in need * coverage.Cost per service (for each intervention) = Number of services * unit costs per service.The total cost is the sum of all intervention costs, plus the costs of the above facility program costs.Total costs = Sum of all costs per services + program costs.We define the full financial cost as the sum of facility-level and above-facility costs , drug and supply costs, other direct costs including nonconsumables and training that take place at the facility, and indirect costs including capital costs such as buildings, support personnel, maintenance/utilities, and supervision/management costs that are specific to the facility .The number of minutes required to deliver a service by provider type is calculated based on the number of visits and time per visit specified in the treatment inputs for each intervention. A cost per minute for each provider type is calculated based on wages and benefits and time utilization.Drug and supply costs are estimated using an ingredients approach, with information on the unit cost, percent of clients receiving, and number of units for each item .Other recurrent and capital costs are applied as a cost per inpatient day and per outpatient visit. The number of inpatient days and visits are multiplied by the cost per day or visit.Above-facility or program costs include all costs at the district, national, or international level that support the service, including program-level human resources, training that takes place above the facility level, supervision, monitoring and evaluation, infrastructure and equipment used in program-level activities, transport, communications, media and outreach, advocacy, and general program management. Each of these elements is explained further below; note that care should be taken to distinguish activities taking place at the facility vs. above-facility levels, especially in the case of training and capital costs, or double-counting could occur. Note further that all of these values can be changed in the software, and in an application should be reviewed and modified as appropriate.For a pictorial representation of the LiST Costing cost items, see Fig. Target populations are drawn from the demographic projection, the AIDS impact model and LiST, including numbers of pregnancies and births, and estimates of children in different age brackets. Populations in need are drawn from LiST inputs and results . Additional populations in need that are not available from a Spectrum impact model are drawn from OneHealth Tool databases, such as the percent of pregnant women needing management of pre-eclampsia.Treatment inputs for each intervention specify the required drugs and consumable supplies , provider time, and number of inpatient days and outpatient visits needed for the effective provision of an intervention. These are drawn from intervention assumptions developed for the OneHealth Tool and documented in an Input Intervention Assumptions Manual . These iDefault treatment inputs and prices are provided at a global level with no variation for different countries, but these inputs can be adjusted to fit the country context. Users can change the assumed quantities of drugs and supplies used, amount of provider time and number of visits, as well as the unit price for any input. In a usual country application, country teams review all data assumptions thoroughly, particularly country-specific input prices, and change the assumptions to reflect the specific country context.Drug pricing defaults are updated every one to two years, depending on price stability for a given commodity. Updates for the drug prices that change more quickly, such as antiretroviral HIV/AIDS drugs, take place on an annual basis. Treatment inputs are updated when the treatment norms change, such as shifts in the drug regimens recommended by WHO, or following the introduction of new therapies. These changes typically take place less frequently and such database updates may occur every 3 years or longer intervals.Default salaries are provided for each country based on WHO CHOICE , but canprogram management, or any activities above the facility level that are utilized in running the program at the district, national, or international level; research, which includes support for programs that collect primary data , as well as data analysis, report writing, and dissemination; monitoring and evaluation (M&E), which includes salaries of M&E officers, equipment for data processing, communications, and transportation as well as the costs of collecting M&E data; communication, media, and outreach, which includes personnel costs for preparing analyses and presentations, conducting awareness-raising and policy dialogue with opinion leaders, policy makers, and stakeholders, office support costs, and transportation and materials production associated with dissemination activities; training, which includes both pre-service and in-service training for health care workers as well as special training for program staff in areas such as strategic planning, M&E, advocacy, and financial and reporting systems; and infrastructure and equipment, which includes costs associated with equipment acquired at the central, or above-facility, level. Many costs relating to improving the quality of care would be managed in this section. An initial suggested list of line items for program costs has been supplied by default but can be edited by the user.The user must supply either the amount of, or the percentage over and above, the facility-level costs associated with various above-facility program support activities, as shown in the second box on the left-hand side of Fig. http://www.who.int/choice/cost-effectiveness/en/). Note that the costs that are available on their website are defined by the World Health Organization to be the “hotel” cost portion of both OPVs and IPDs, that is, all costs except drugs and laboratory costs. In other words, the OPV and IPD costs contain both other direct costs (ODCs) and indirect costs, as well as personnel costs and the cost of consumables.The costs per outpatient visit and inpatient day (OPVs/IPDs) have been calculated by the World Health Organization at the country level and are available from the WHO-CHOICE website . Although it would have been ideal to utilize a dataset encompassing all MNCH interventions in LiST in order to perform this estimation, unfortunately such a dataset does not exist. Instead, we use the VMMC dataset which contains complete, consistent, and disaggregated facility-level data across multiple facilities and countries , and which has been used extensively and described in peer-reviewed literature –21. The We estimate a four-factor cost function comprised of Personnel, Consumables (excluding drugs and laboratory costs), Other Direct Costs (including nonconsumables and training), and Indirect Costs . We excluded drugs and laboratory costs from the Consumables variable in the VMMC dataset, where they were initially included, in order to mirror the “hotel” cost definition of the OPV/IPD cost in the WHO-CHOICE dataset. We estimated a factor shares equation in Stata using a translog cost function, correcting the standard errors for heteroscedasticity using the Huber/White/sandwich estimator .LiST costing utilizes the coverage levels specified by users in the standard LiST editors. This coverage is utilized as part of the equation to establish number of services, as detailed in the methods section of this article. This ensures consistency between the cost and impact calculations of the tool.Several tools exist to facilitate strategic planning for MNCH programs, including LiST Costing, the OneHealth Tool, and EQUIST. Users should select from among them based on their analysis questions and purposes for the information being generated.LiST costing is designed to help users prioritize from among a core set of MNCH interventions. It is automatically linked with LiST impact calculations and incorporates additional costing databases, such as wages, which allows for relatively quick generation of results to allow program planners and policy makers to think through the potential cost and resources required for implementing different packages of services.The OneHealth Tool is designed for holistic health sector costing and budgeting and includes a broad suite of costing and impact analysis modules, including MNCH , but also HIV, TB, NCDs, malaria, and others. It requires more data collection and entry by the user around the health system costs such as wages, and infrastructure costs such as construction and operation of facilities. It provides detailed costing templates around the cost categories that are aggregated into other recurrent costs, capital cost, and program costs in LiST costing . This tool is typically used when estimating cost and impact of a whole health sector plan. Users who are focused more on MNCH interventions, and don’t need to have detailed outputs on health systems components will likely use LiST costing, while the OneHealth Tool will be preferred by users looking at a broader set of interventions and/or health systems implications of a plan.EQUIST is a web-based tool designed to help policy makers identify strategies and approaches to reduce health disparities between advantaged and disadvantaged groups , and to examine the health outcomes of achieving greater parity. For more detail on different tools which include MNCH costing, see Table Researchers and program planners should think through their research question and select the appropriate tool accordingly. Complementarity does exist between these tools: LiST costing is in many ways a streamlined and MNCH-focused component of the OneHealth Tool, and EQUIST uses LiST for its impact calculations. However, none are identical in their outputs and approach to analysis, so it is worth taking the time to think through the scope and specific goals which users are trying to achieve and select accordingly.Limitations include using VMMC data in the estimation of ODCs and indirect cost proportions for a maternal and child health model; ideally a comprehensive, consistent dataset specific to MNCH would be utilized, but to our knowledge none are available.Moving forward, one area of further development would be to facilitate the ease of regional applications. As health systems become more and more decentralized, increasingly planning will take place at a sub-national level. One suggestion is to provide a way to link a preset data input form directly with LiST, so that regional-level data can be utilized more easily.Achieving the Sustainable Development Goals requires choices to be made for investment in cost-effective interventions. Here, we describe the methods, data and results of the LiST Costing module which allows countries and researchers to calculate both the full financial costs and the impacts of MNCH intervention scale up on morbidity and mortality. It relies on existing intervention inputs and cost databases as much possible to ensure consistency with other models which countries may be using, while providing a more streamlined and simpler platform for estimates of program costs. As donors and policy makers consider the range of options available for health investment, evidence-based analysis of different resource allocation strategies is crucial, and LiST costing provides a user-friendly tool to consider cost effectiveness as a factor in these decisions.Additional file 1:List of interventions included in LiST Costing. (DOCX 14 kb)Additional file 2:Default drug and supply costs for selected services. (DOCX 17 kb)Additional file 3:Cost functions. (DOCX 38 kb)


N,O-carboxymethyl chitosan (NOCC) solutions in aqueous media containing calcium chloride. To prepare drug-loaded beads, sulfasalazine (SA) as a model drug was added to the initial aqueous polymer solution. These beads were characterized and evaluated in vitro as potential carriers for colon-specific drug delivery. A 32 full factorial experimental design was employed to evaluate the effect of polymer and CaCl2 concentrations on swelling and drug release behavior of the beads in simulated gastrointestinal tract fluid. It was found that the rate of swelling and drug release decreased significantly with increasing polymer and CaCl2 concentrations, but polymer concentration was more effective than CaCl2 concentration. The beads prepared using 4.5% polymer concentration and 4% CaCl2 concentration retained approximately 60% of the loaded drug before approaching the simulated colonic fluid. Based on the results, the alginate-NOCC beads prepared with high polymer concentration could be potentially suitable polymeric carriers for colon-specific delivery of SA.In this study, pH-sensitive blended polymeric beads were prepared by ionic gelation of mixed alginate and The online version of this article (doi:10.1186/2194-0517-2-10) contains supplementary material, which is available to authorized users. The use of polysaccharides in the formulation of colon-specific drug delivery carriers has gained increasing interest lately  is a chitosan derivative bearing a carboxymethyl substituent at some of the amino and primary hydroxyl sites of the glucosamine units of the chitosan structure. Biodegradability, biocompatibility, excellent water solubility, gel formation ability, and amphoteric polyelectrolyte characteristics make this material suitable for biomedical applications  release from these carriers in simulated gastrointestinal fluid.In the present study, a 35) with an 85% degree of deacetylation was provided from Sigma-Aldrich Corporation . Sodium alginate was obtained from BDH Laboratory . Calcium chloride, monochloroacetic acid and isopropyl alcohol were purchased from Merck . Sulfasalazine was obtained from Zhejlang Jiuzhou Pharmaceutical Co. Ltd . NOCC was synthesized according to the literature , were prepared separately. Next, equal volumes of these solutions were mixed to form a homogenous blend solution which was maintained for 5 h for the complete removal of bubbles. The final pH of the solution was found to be approximately 7.5 ± 0.1. Five milliliters of these solutions was dropped into a 30-ml gently stirred CaCl2 solution with distinct concentrations of 1%, 2.5%, and 4% (w/v) through a syringe needle (0.4 mm in diameter) at a dropping rate of 1.0 ml/min. The distance of the needle tip from the gelling solution surface was 10 cm. The prepared beads were allowed to harden in the calcium chloride solution for 30 min. These beads were filtered, washed with distilled water three times, and dried at 40°C for 24 h or freeze-dried. The freeze-dried beads were obtained through rapid freezing at −80°C, followed by drying in a freeze drier .Firstly, aqueous alginate and NOCC solutions, with concentrations of 1.5%, 3%, and 4.5% (w/v) was added to the initial aqueous alginate solution with continuous stirring, and the pH of the solution was adjusted to 7.5 by adding 2 M NaOH. This solution was used for the preparation of SA-loaded beads by the same procedure described for the preparation of unloaded counterparts.To prepare drug-loaded beads, SA with a final concentration of 1%  was calculated from the difference between the amount of SA dissolved in aqueous polymer solution and that of SA released in gelation medium divided by the amount of SA dissolved in aqueous polymer solution. For this purpose, the concentration of SA in gelation and washing solution was determined spectrophotometrically at 359 nm. Drug content (wt.%) was determined as the ratio of encapsulated SA weight to the total weight of the dried beads. This was accomplished by immersion of drug-loaded beads in sodium phosphate buffer at pH 7.4. The total released drug after 24 h was determined spectrophotometrically and was considered as encapsulated SA.Φ(τ), at each time was calculated using the following expression:The swelling characteristics of beads were determined by immersing them in dry state into conical flask containing 40 ml of release medium that were incubated at 37°C under shaking at 150 rpm. At first, the dry beads were swollen in 0.1-M HCl solution at pH 1.2 (simulated gastric fluid) for 2 h. Afterwards, the beads were transferred to a sodium phosphate buffer solution at pH 6.8  and kept for 3 h. Subsequently, they were transferred to a sodium phosphate buffer solution at pH 7.4 (simulated colonic fluid) until complete dissolution was obtained. At specific time intervals, samples were taken out from the swelling medium and blotted with a piece of paper towel to absorb excess water on the surface. The degree of swelling, Ψτ and Ψ0 are the sample weights at time τ and in the dry state, respectively. Each experiment was repeated three times.where The SA release from drug-loaded beads was studied using the same conditions as described in the swelling studies. At predetermined time intervals, 2 ml of samples were withdrawn from the dissolution medium and immediately replaced by the same volume of fresh medium. The amount of SA released from the beads was determined spectrophotometrically  at 359 nm using previously calibrated standard curves at different pH values. To determine the release in 0.1-M HCl solution, the pH of the release medium was adjusted to 7.4 by adding NaOH, and the concentration of SA was determined from the calibration curve at this pH. Each experiment was repeated three times.A full factorial design with two parameters at three levels, as shown in Table In our previous study  on NOCC or alginate, established by Ca2+. Thus, after the dropping of mixed alginate-NOCC solution into calcium chloride solution, alginate entangled through the NOCC network and vice versa, resulting in the formation of interpenetrating polymeric network. Zhang et al.  polymer concentration, the spherical shape of the beads changed to an irregular shape with a collapsed center and some cracks on the surface ) remained almost spherical with a rather rough surface and compact structure.The photographs of wet, freeze-dried, and air-dried beads, taken under an optical microscope, are shown in Figure e Figure c. The beThe swelling behavior of alginate and alginate-NOCC beads in simulated gastrointestinal fluid is shown in Figures At pH 1.2, the swelling of alginate and alginate-NOCC beads was hindered due to the formation of strong hydrogen bonds between -COOH and -OH groups of both polymer polar chains , due to increased cross-linking density of the network. At these media, the swelling and disintegration rate decreased significantly with increasing polymer concentration (p < 0.05). This may be related to increased elastic force that resulted from (1) hydrogen bonding between amine and hydroxyl groups of NOCC and alginate, (2) electrostatic attraction between ionized amine and carboxyl groups of NOCC and alginate, and (3) physical cross-linking as a result of polymer chain entanglements.At neutral and basic media, the swelling degree of alginate-NOCC beads, prepared at constant polymer concentration, decreased with increasing CaClComparing Figures 2 concentrations is shown in Figure p < 0.05) in accordance with the swelling behavior of beads  is given in Table Analysis of variance, performed by Minitab 14 software, for the effect of polymer and CaCl2 concentration. According to the interaction plot of these factors presented in Figure 2 concentration increasing from 2.5% to 4% became smaller at higher polymer concentrations. This can be related to the formation of densely cross-linked polymeric layer on the surface of droplets which resists to Ca2+ diffusion into the beads' core, leading to the formation of beads with unreacted or partially reacted core with smaller resistance to swelling and drug release.As shown in the main effect plot presented in Figure 2 concentration of 4% or 2.5% retained approximately 60% of loaded drug before exposure to the simulated colonic fluid. This is a promising property for the application of optimized alginate-N,O-carboxymethyl chitosan gel beads as colon-specific delivery system.As shown in Figure 2 concentrations. Therefore, the beads can be further evaluated for the release of drugs in the different segments of the gastrointestinal tract. This strategy is more convenient than the coating procedure used in a previous study (Tavakol et al. The swelling and drug release characteristics of the alginate-NOCC beads can be tuned by the modulation of polymer and CaCl2 concentrations, but polymer concentration was more effective. No burst effect was observed for SA release from these pH-sensitive carriers. It was previously shown that the drug release behavior of SA-loaded alginate-NOCC beads was improved by chitosan coating (Tavakol et al. Calcium cross-linked alginate-NOCC beads prepared in the present study demonstrated distinct pH-sensitive swelling and drug release behavior. Sulfasalazine release rate was slow in acidic medium but increased at pH 6.8 and pH 7.4, which is in accordance with the swelling rate of the beads and SA solubility. The rate of SA release decreased with increasing polymer and CaClMT is a PhD student. EVF is a professor and supervisor. SHN is an assistant professor and co-supervisor.