Adenovirus ocular infections are common ocular viral infections seen worldwide, for which there is no approved antiviral therapy available. Ranpirnase is a novel ribonuclease which preferentially degrades tRNA resulting in an inhibition of protein synthesis. The study goal was to determine the anti-adenoviral activity of topical formulations of ranpirnase (OKG-0301) on adenoviral replication in the Ad5/NZW rabbit ocular replication model. NZW rabbits were inoculated in both eyes with human adenovirus type 5 (HAdV5) after corneal scarification. A day later, topical therapy was initiated in both eyes with 0.03% OKG-0301, 0.003% OKG-0301, saline or 0.5% cidofovir. Eyes were cultured to determine HAdV5 eye titers over 2 weeks. OKG-0301 (0.03% and 0.003%) and 0.5% cidofovir decreased viral titers compared to saline. Furthermore, both OKG-0301 formulations and 0.5% cidofovir shortened the duration of the HAdV5 infection compared to saline. Both 0.03% OKG-0301 and 0.003% OKG-0301 demonstrated increased antiviral activity compared to saline in the Ad5/NZW rabbit ocular replication model. The antiviral activity of the OKG-0301 groups was similar to that of the positive antiviral control, 0.5% cidofovir. Ranpirnase (OKG-0301) may be a potential candidate for a topical antiviral for adenoviral eye infections. Further clinical development is warranted. Human adenoviruses (HAdV) are the etiological agents of the most common ocular viral infections worldwide, accounting for up to 75% of all conjunctivitis cases . ApproxiPharyngeal conjunctival fever is a less severe form of adenoviral ocular infections that is seen mostly in children . This syCurrently, there is no approved antiviral for the treatment of these infections. An effective antiviral agent for adenoviral ocular infections would fulfill an unmet medical need in ophthalmology. It would reduce patient pain and suffering during the acute infection and by decreasing the amount of virus on the eye, the formation of the vision-altering subepithelial infiltrates can be reduced or eliminated.Several antiviral categories and strategies have been evaluated as potential treatments of HAdV eye infections. Nucleoside analogs ,3,4,5,6,Rana pipiens,  which belongs to the RNase A superfamily [A new approach to antiviral development is the use of ribonucleases, enzymes that degrade RNA. Ranpirnase is a ribonuclease that is extracted from the oocytes of Northern leopard frogs erfamily . Ranpirnerfamily .Ranpirnase is a small enzyme (11.82 kDa). Its active site contains the catalytic triad  that is characteristic of the RNase A superfamily . HoweverRanpirnase has been extensively studied for its antitumor activity for which it has a unique mechanism of action. It is a biological drug that is administered extracellularly but acts intracellularly; therefore, it must be internalized into the cell . RanpirnIn contrast, much less is known about its mechanism of action as an antiviral. It has been speculated that its antiviral mechanism of action is similar to its anti-tumor activity in that the RNA interference results in direct virus inhibition, degradation of viral RNA, disruption of TNF-α signaling, and changes in host-cell gene expression ,14. RanpIt has been speculated that ranpirnase may have antiviral activity against adenovirus infections. This prompted this study where the goal was to evaluate the anti-adenovirus activity of topical ocular formulations of ranpirnase, heretofore known as OKG-0301, in the Ad5/NZW rabbit ocular replication model. We have used this model to evaluate the effect of several antivirals on adenovirus replication in vivo ,10,11.0.03% and 0.003% OKG-0301 were prepared in intravenous saline from 1 mg vials of ranpirnase that were provided by Okogen, Pty Ltd., Melbourne, VIC, AUS. Cidofovir 0.5% (CDV) was diluted in intravenous saline from the 7.5% injectable form of cidofovir  and functioned as the positive antiviral control. Intravenous saline  functioned as the diluent for the experimental antivirals and as the negative control (CON).50 in 0.1 mL of the virus isolate was separately mixed for 1 h with 0.1 mL of several dilutions  of antisera of several adenovirus types purchased from ATCC . After incubation, the virus-antibody samples were inoculated onto separate wells of a 96-well plate containing A549 human lung carcinoma cells . The plates were incubated at 37 °C, in 5% CO2 for up to 10 days. Cells that were treated with the virus-antibody mixture that did not demonstrate cytopathic effect (CPE) denoted to which adenovirus type the isolate belonged.The ocular HAdV5 strain used in this study was recovered from a patient with typical adenoviral ocular disease at the Charles T. Campbell Ophthalmic Microbiology Laboratory at the UPMC Eye Center, Department of Ophthalmology, University of Pittsburgh School of Medicine. It was determined to be adenovirus type 5 (HAdV5) using a standard serum neutralization technique . BrieflyThe use of this de-identified isolate did not require an Institution Review Board (IRB)/Ethics committee approval because the patient contact or personal information was not compromised.A549 human lung carcinoma cells (RRID:CVCL_0023) (ATCC) were used in this study.2. When the cells exhibited 100% CPE (1–3 days), the cells were scraped from the flask and the media plus cells were transferred to a 50 mL polypropylene conical tube. The cells were centrifuged for 5 min at 1800 RPM at 4 °C. The supernatant was discarded. The pellet was resuspended in media. Three cycles of freeze/thaw/vortex are preformed using liquid N2 and a 37 °C water bath. The virus was then aliquoted and stored in a −80 °C freezer until use. The titer of the HAdV5 stock was determined as described in The stock of the HAdV5 isolate was prepared in A549 cells. Briefly, the cells were grown to 85–95% confluence in tissue culture media consisting of 1X Minimum Essential Medium  with 1% Gentamicin Solution (50 mg/mL) , 10% Penicillin-Streptomycin (5000 U/mL) (Gibco), 25 µg/mL Amphotericin B (Sigma-Aldrich), and 10% Fetal Bovine Serum . The media were removed from the A549 cells, then the cells were inoculated with a small volume of HAdV5 at a multiplicity of infection (MOI) of 5 to 10. Following a 3-h adsorption period, the inoculum was removed, and media were added to the cells. The cells were incubated at 37 °C, in 5% COHAdV5 was used in this study, as it was in previous ocular adenovirus antiviral studies ,10,11. INew Zealand White female rabbits weighing 1.1–1.4 kg (approximately 6 weeks old) were obtained from the Oakwood Research Facility through Charles River Laboratories, Wilmington, MA. This study conformed to the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) approval (Protocol #16027750) was granted, and US Animal Welfare Act guidelines were observed. The authors confirm that this study complied with the ARRIVE guidelines.6 PFU/eye) of HAdV5 in both eyes. Rabbits were treated with intramuscular injections of ketoprofen, 1.5 mg/kg on the day of inoculation and the next day. The following morning, the rabbits were randomly allocated to one of four topical treatment groups: (1) 0.03% OKG-0301—8X/day for 9 days (n = 9); (2) 0.003% OKG-0301—8X/day for 9 days (n = 10); (3) Saline—8X/day for 9 days (n = 10); and 0.5% CDV—2X/day for 7 days (n = 9). Rabbits were topically treated in both eyes according to the regimens described above. Cidofovir was administered only twice daily for 7 days as this is our standard treatment regimen using this agent [This experiment was carried out using duplicate trials resulting in a total of a total of 38 rabbits. After general anesthesia with intramuscular injections of ketamine 40 mg/kg) and xylazine (4 mg/kg) was achieved, topical anesthesia was produced with 2 drops of 0.5% proparacaine. The rabbits were the transferred to a table fitted with an operating microscope where, 12 cross-hatched strokes of a #25 sterile needle were performed on both corneas. The rabbits were then inoculated with 50 µL  was based on previous studies ,4,5,7,10 was base2, the media were aspirated from the wells and the cells were stained with 0.5% gentian violet in formalin. The plates were dried and the numbers of HAdV5 plaques per well were counted using a dissecting microscope. The viral titers for each sample were calculated and presented as plaque forming units per milliliter (PFU/mL).The eye cultures to be quantified were thawed, diluted, and 0.1 mL of the undiluted and diluted samples were inoculated in duplicate onto A549 cell monolayers contained in 24-well multiplates. The viral inoculum was adsorbed for 3 h after which 1 mL of tissue culture media containing 0.5% methylcellulose was added to the wells. After 7 days of incubation at 37 °C in 5% COp ≤ 0.05.The HAdV5 titer data were analyzed using Kruskal–Wallis ANOVA with Duncan’s multiple comparisons and Fisher’s Exact Test . Significance was defined as This antiviral study was conducted using the Ad5/NZW rabbit ocular replication model. This model evaluates ocular HAdV5 replication only. Signs of infection in the eyes are not produced in this model and therefore were not assessed. Ocular toxicity also was not evaluated in this model because of the corneal scarifications during the viral inoculations and the slight ocular trauma induced by the act of culturing the eyes. This is not a true representation of ocular toxicity that would be produced in naïve rabbit eyes. The ocular HAdV5 titer data were analyzed to determine the percentage of HAdV5 positive cultures per total for each culture day, the HAdV5 titers for each culture day, and the duration of shedding which is the length of the HAdV5 infection in the eyes.p ≤ 0.0208), while 0.003% OKG-0301 significantly decreased the percentage of HAdV5 positive cultures per total cultures compared to CON on Day 11 only (p = 0.0156). The positive antiviral control, 0.5% CDV significantly decreased the percentage of HAdV5 positive cultures per total cultures on Days 5, 7, and 11 compared to CON (p ≤ 0.0208). In addition, treatment with 0.03% OKG-0301 significantly decreased the percentage of HAdV5 positive cultures per total cultures on Day 7 compared to treatment with 0.003% OKG-0301 (p = 0.0076).The most rigorous of the viral outcome measures is the percentage of HAdV5 positive cultures per total for each day that was assessed. Any sample with a detectable HAdV5 titer was deemed to be an HAdV5 positive culture. These data were analyzed using the Fisher Exact Test (FET) . Treatme10 median ± interquartile ranges . The data are expressed as Loge ranges . All antp ≤ 0.05, K-W). In addition, 0.03% OKG-0301 and 0.5% CDV significantly shortened the duration of HAdV5 shedding compared to 0.003% OKG-0301 . There was no significant difference between 0.5% CDV and 0.03% OKG-0301.The final days on which eyes had positive HAdV5 cultures were used to determine the duration of HAdV5 shedding. This viral outcome measure is shown as the median (±interquartile ranges) length of the infection in the eyes. Treatment with 0.03% OKG-0301 (5.0 ± 2.00 days), 0.003% OKG-0301 (7.0 ± 0.00 days), and 0.5% CDV (7.0 ± 2.75 days) significantly decreased the length of infection compared to the saline control (11.0 ± 4.00 days) (Ribonucleases (RNases) are enzymes that cleave RNA. These enzymes play a key role in the development of mRNA and noncoding RNA, the operation of RNA interference, and the control of gene expression in all organisms . A numbeRanpirnase has demonstrated antiviral activity against rabies viruses , HIV 1515,16, HPThe demonstration of antiviral activity for ranpirnase against both RNA and DNA viruses led us to the current study for which we determined the antiviral activity of topical formulations of ranpirnase in an experimental model of an ocular adenovirus infection. We have used this model to evaluate a number of potential antiviral agents for the treatment of adenovirus ocular infections ,7,10,11.In the current study, both 0.03% and 0.003% OKG-0301 demonstrated antiviral activity against adenovirus in the Ad5/NZW rabbit ocular replication model. Both OKG-0301 formulations produced significant decreases in the percentage of HAdV5 positive cultures , decreases in daily HAdV5 ocular titers , and in the median duration of ocular HAdV5 shedding compared to the negative control . These results are noteworthy because decreasing the adenovirus ocular titers and shortening the length of the infection can lead to a decrease in the morbidity associated with these infection as well as a possible reduction or prevention of the formation of vision altering subepithelial corneal infiltrates that are characteristic of adenovirus eye infections.There were several concentration-dependent differences in viral outcome measures between the two OKG-0301 concentrations. The 0.03% OKG-0301 concentration significantly reduced the median duration of shedding by 2 days compared with 0.003% OKG-0301 and reduced the percentage of HAdV5 cultures and titers on Day 7.To summarize this initial experimental study, two concentrations of an ocular formulation of ranpirnase, OKG-0301, demonstrated significant antiviral activity compared with saline in the Ad5/NZW rabbit ocular replication model. The antiviral efficacy demonstrated by the higher ranpirnase concentration was comparable to the positive antiviral control, 0.5% cidofovir, while the lower concentration was slightly less efficacious. There was a difference in antiviral efficacy between the two ranpirnase concentrations suggesting concentration-dependent antiviral activity.As mentioned above, the goal of the current study was to only evaluate the antiviral activity of topical ranpirnase. The ocular toxicity of topical ranpirnase was not evaluated in this study. Subsequent to this experimental study, a Phase 2 clinical trial in patients with adenoviral conjunctivitis was initiated (NCT03856645) . PrelimiIn conclusion, ranpirnase demonstrated antiviral activity against an experimental ocular adenovirus infection in the Ad5/NZW rabbit ocular replication model. Based on the results of this study, further development of ranpirnase as an antiviral for the treatment of adenovirus ocular infections in patients is indicated.


Crossbred steers were utilized in the growing  and finishing  experiments, blocked by BW, and assigned randomly to 16 pens. Pens were assigned randomly to one of two treatments (biochar vs. control) with eight replications per treatment. Four pen replications per treatment were paired within BW block and rotated randomly through an emissions barn with two chambers  to capture average weekly emissions of CH4 and CO2. Pen was the experimental unit and chamber was included as a fixed effect for emissions data. There were no statistical differences (P ≥ 0.23) in performance outcomes between treatments for the growing experiment. Dry matter intake  and average daily gain  were 2.2% and 5.9% lower for biochar-fed steers in the finishing experiment, respectively, resulting in a lighter hot carcass weight (P = 0.10) and lower calculated USDA yield grade (P = 0.02). Emissions of CH4 and CO2 were not affected by biochar inclusion in the growing (P ≥ 0.22) or finishing experiment (P ≥ 0.60). Results from these experiments show no indication that feeding biochar, supplemented at 0.8% (growing), and 1.0% (finishing) of the diet DM, reduces methane emissions in growing or finishing cattle.A feedlot growing (77-d) and finishing (111-d) experiment was conducted to evaluate the effects of feeding biochar on steer performance, methane and carbon dioxide emissions, and carcass characteristics. Two treatments were evaluated, a control diet without biochar and the same diet with biochar included at 0.8% of dietary DM (growing) or 1.0% of dietary DM (finishing). The growing diet consisted of 40% corn silage, 40% wheat straw, 15% modified distillers grains plus solubles, and 5% supplement, with 0.8% biochar replacing fine ground corn in supplement. The finishing diet consisted of 55% high-moisture corn (HMC), 35% Sweet Bran, 5% wheat straw, and 5% supplement, with biochar replacing 1.0% HMC and added as an ingredient. Biochar was sourced from ponderosa pine wood waste  and was 83% C with 426 m Methane is emitted to the atmosphere via natural sources, such as wetlands and enteric fermentation from wildlife, and by human activities, including the petroleum and natural gas sectors and enteric fermentation from domesticated ruminant animals  at the 0.8% inclusion rate in the growing and finishing experiments, respectively. Biochar utilized in 3, and surface area of 323 m2/g.One proposed method to reduce CHf oxygen , resultipulation . When bipulation  and barlpulation , a reducobserved . Previou4 production (g/kg DMI). There are a broad variety of biochars available, and the characterization of the product can differ significantly . Biochar is not currently approved by the U. S. Food and Drug Administration (FDA) to be fed to cattle intended for human consumption. Prior to experiment initiation, a food use authorization from the FDA was obtained for cattle utilized in this experiment to enter the human food chain.4 emissions from cattle. Biochar utilized for both the growing and finishing experiments was provided by High Plains Biochar LLC  and was sourced from ponderosa pine trees as part of forest wood waste. Biochar was processed by High Plains Biochar LLC to a small particle size to reduce opportunity of cattle sorting in the bunk. Six randomized grab samples of biochar collected through the growing and finishing periods were sent to Control Laboratories  for chemical analysis. Dry matter of the biochar fluctuated with ambient moisture from 57% to 76% DM with an average of 70%. On a DM basis, biochar carbon content was 82.8%, with a surface area of 426 m2/g, bulk density of 107.8 kg/m3, total N content of 0.7% of total dry mass, and pH of 9.49. Biochar particle size distribution ranged from less than 0.5 mm to 8 mm, with approximately 66% of sampled biochar measuring less than 2 mm, and 1% of sampled biochar measuring greater than 4 mm.A 77-d feedlot growing experiment was conducted at the University of Nebraska-Lincoln (UNL) Eastern Nebraska Research, Education, and Extension Center (ENREEC) near Mead, NE, to evaluate the impact of biochar inclusion in a forage-based diet on performance and CHn = 160; initial BW = 363 kg; SD = 16 kg) were assigned to two treatments . Pens were assigned randomly to treatment (n = 16).Steers , and an injectable solution for the treatment and control of gastrointestinal roundworms, lungworms, eyeworms, lice, and mites . Before experiment initiation, steers were limit-fed a common diet of 50% alfalfa hay and 50% Sweet Bran  offered at 2% of BW for 5 d to equalize gut fill , a killed vaccine for clostridial toxoids and gut fill . Steers n = 128; initial BW = 480 kg; SD = 17 kg) remained in the same treatment groups as the growing experiment, and 2 steers (lightest BW) were removed from each pen to better accommodate chamber space in the emission barn, reducing pen count from 10 to 8 steers per pen. Two treatments were evaluated in the finishing experiment , and neutral detergent fiber (NDF) content. Feed samples  were oven-dried at 60 °C for 48 h  and CP (Cattle were adapted to the finishing diet in 4 steps over 21 d. Step 1 diets were fed from day 1 to day 5 and contained (DM-basis) 35% Sweet Bran, 31% wheat straw, 29% HMC, and 5% supplement. Step 2 diets were fed from day 6 to day 11 and contained 35% Sweet Bran, 24% wheat straw, 36% HMC, and 5% supplement. Step 3 diets were fed from day 12 to day 16 and contained 35% Sweet Bran, 17% wheat straw, 43% HMC, and 5% supplement. Step 4 diets were fed from day 17 to day 21 and contained 35% Sweet Bran, 10% wheat straw, 50% HMC, and 5% supplement. Biochar at 1.0% of the dietary DM replaced HMC in each of the above steps for the biochar treatment.2), where KPH fat was assumed to average 2.5%. Carcass adjusted final BW was calculated from HCW divided by a common dressing percentage of 63.Steers were harvested at a commercial abattoir  at experiment completion. On the day of shipping, pens were offered 50% of the previous day’s feed offering at regular time of feeding. Cattle were loaded and shipped to the abattoir in the afternoon for slaughter the next morning. Hot carcass weights (HCW) were recorded on day of slaughter and USDA marbling scores, 12th rib fat thickness, and longissimus muscle (LM) area were recorded after a 48-hr chill. Calculated yield grade was determined using the following equation : 2.50 + 4 and CO2 production, was utilized for 8 consecutive weeks to monitor emissions from growing steers, followed by an additional 8 consecutive weeks to monitor emissions from finishing steers. The emission barn, as described by 2 and CH4, respectively. Eight pens of cattle, 4 control, and 4 biochar, were selected randomly to rotate through the methane barn by pairing replications within BW block, representing 1, 2, and 1 rep from light, middle, and heavy block, respectively. Pairings were rotated through the barn for two 5-d periods, with each treatment represented in the barn concurrently. Each week, steers entered the barn Wednesday morning and remained in the barn until Monday morning when they were returned to their respective feedlot pen. Manure CO2 and CH4 emissions were measured from the accumulation of 5 d of manure buildup and was calculated for the remainder of Monday when cattle were absent from barn. The barns were scraped clean using a skid steer each Tuesday to develop a baseline emission level post manure removal. Baseline emission levels of CO2 and CH4 were subtracted from manure emission levels of CO2 and CH4 and final values were divided over 5 d and 10 steers (growing experiment) or 8 steers (finishing experiment), to account for individual animal emissions. Following these steps, average CO2 and CH4 values of 16.89 g and 0.08 g per steer, respectively, were subtracted from the daily emissions for CO2 and CH4 in the growing experiment due to manure emissions, and average CO2 and CH4 values of 17.45 g and 0.07 g per steer, respectively, were subtracted from the daily emissions for CO2 and CH4 in the finishing experiment. Gas emissions of CH4 and CO2 were reported as g/steer and g/kg of DMI, where reported DMI (kg/d) used for the gas emission calculations were averaged from the weekly intakes of each treatment during rotation through the respective emission chambers.The UNL ENREEC emission barn, equipped with a negative pressure system to monitor and record CH2 and CH4 reached greater than 60 ppm at certain points throughout the day, which may be beyond the capacity of the sensor for accurate measurement  had unusable data for CH4 emissions. Significance was considered at α ≤ 0.05 and a tendency was considered at 0.05 < α ≤ 0.10.Performance and emissions data were analyzed using the MIXED procedure of SAS  with pen as the experimental unit. Performance data included BW block as a fixed effect. For emissions data in the growing experiment, day was a repeated measure. During the growing experiment, 6 d  were not usable for emissions measurement due to complications with the barn sensor recording. Concentrations of COsurement . UnexpecP = 0.46), dry matter intake , or feed efficiency (P = 0.25), between biochar supplemented steers and control , type of cattle, and differences in measurement techniques may all contribute to the differing results.There were no statistical differences in performance outcomes, including average daily gain  and average daily gain  and tended to have a lighter carcass adjusted final BW (P = 0.10) compared to the control (P = 0.22). The significant reduction in DMI observed for biochar-fed steers in the finishing experiment is dissimilar to results in previous literature  with numerically reduced 12th rib fat (P = 0.12), resulting in a significantly lower USDA calculated yield grade (P = 0.02) compared to the control. Reduced HCW and improved yield were a function of the significant reduction in DMI and ADG for biochar-fed steers compared to the control steers. A Canadian experiment by P ≥ 0.93) which was congruent with Biochar-fed steers tended to be lighter in hot carcass weight  in the forage based growing experiment compared to the concentrate based finishing experiment. However, the 2 experiments were not statistically compared. 4 production as around 6% of gross energy intake (GEI) for forage-fed cattle and 3.5% for concentrate-fed cattle. 4 production from cattle native to Southeast Asia when supplemented rice husk biochar at 0.62% of a high-forage (cassava root chip) diet. 4 production (g/kg DMI) with pine-sourced biochar inclusion of 0.8% in high-forage and high-concentrate diets, respectively. Headbox experiments are usually very accurate, but can be limited in scope of reference , have greater enteric CHeference . Althoug4 production is based on one or more of the following theories: biochar adsorbs ruminal CH4 gas, biochar increases the inert surface area of the rumen resulting in greater opportunity for microbial colonization, and biochar alters the rumen microbial community (4 in the rumen is no longer the leading theory accepted in literature, considering that previous experiments reporting reductions in enteric CH4 production included biochar at <1.0% dietary DM (The mechanism by which dietary biochar inclusion reduces enteric CHommunity , 2013. Tetary DM , which ietary DM . Improveetary DM .4 emissions. The CH4 emissions findings from 3, and pH of 10.5. Type of diet, physical properties of the biochar, and inclusion percentage of biochar in the diet are all potential reasons for differing emission and performance results between experiments.Dissimilar to the findings of 4 emissions; however, during the finishing experiment, biochar inclusion at 1.0% diet DM reduced DMI and ADG, resulting in a tendency for reduced HCW and a significant improvement in lean carcass yield grade, with no impact on CH4 emissions.In conclusion, pine-sourced biochar included at 0.8% of diet DM during the growing experiment did not impact cattle performance or CH


The replacement of 50% of GNC nitrogen with GM and densification of diet had no effect (P>0.05) on average DMI (g), antioxidant status, progesterone concentration, conception rate, non return rate, no of matings per service, lambing rate, body weights of lambs at birth, 15 and 30 days of age. GNC can be partially replaced by GM in the ewes diet without any adverse effect on reproductive parameters, antioxidant status, progesterone concentration and weight of lambs.Guar meal (GM) can be considered as an alternative protein source for the livestock and has a potential value as a feed to animals with a high protein content ranging between 45–55%. A study was conducted to evaluate the effect of partial replacement of groundnut cake (GNC) with guar meal (GM) on reproductive efficiency, hormone profile, antioxidant status in Deccani ewes fed sorghum stover based complete feed blocks (SSCFB). Twenty-one non-pregnant Deccani ewes  were randomly divided into three groups to contain 7 animals each in a completely randomized design. Three dietary treatments viz. S1: conventional feeding (sorghum stover and concentrate mixture fed separately in 50:50 ratios to meet the requirement), S2: SSCFB with GNC as protein supplement in concentrate mixture and S3: SSCFB with GM replacing GNC nitrogen of S2 at 50% level. The ewes were synchronised for estrus with progesterone impregnated intravaginal sponges and naturally mated. The ewes were fed at the rate of 3.1 and 3.7% of their body weight from 1 Effective utilization of available feed resources is the key for economical livestock rearing. The utilization of crop residues for increased animal production has received greater research attention within the past few decades because of the higher quantities of crop residues production. The crop residues predominantly roughages, however are of inferior quality, but owing to several constraints, developing countries like India even today have to depend largely upon these crop residues for feeding ruminant population. A possible and perhaps the most viable proposition could be the complete feed block (CFB) technology which would enable the livestock farmer to utilize these resources more effectively resulting in better performance of the animals. The complete feed blocks provide optimum protein and energy apart from supplying adequate amount of minerals and vitamins for the animals, thus enhances reproductive efficiency. The CFB technology also provides an opportunity for the incorporation of unconventional feed ingredients such as guar meal in diets.Cyamopsis tetragonoloba) can serve as a promising alternative source of protein for the animal feed industry. Guar meal (GM) after gum extraction is a potential source of protein and contains about 40–52% crude protein  wi. (1979) . The pro. (1978)  using ELet al. (1991) [The feed samples were subjected to proximate analysis and fibre fractions as per the methods described by AOAC. (1997)  and Van . (1991) .The experiment was carried out at the College of Veterinary Science, PVNR Telangana Veterinary University, Rajendranagar, Hyderabad  in India. The ambient temperature and relative humidity values during the period of study were in the range of 31–38°C and 28–39%, respectively. The experimental protocols were done with the approval of Institute’s Animal Ethics Committee (IAEC) of PVNR Telangana Veterinary University, Hyderabad, 500 030, Telangana, India.www.socscistatistics.com) with significance at 5% level, in a 2×3 contingency table.The results obtained in four phases of experiments were subjected to analysis through software Version 15.0; SPSS) by applying one-way and two way multivariant analysis of variance through generalized linear model (antioxidant enzyme activity and serum progesterone concentration). The treatment means were ranked using Duncan’s multiple range test with significance at 5% level . All the.0; SPSS st to 4th fortnight. By the time of 10th fortnight ewes fed S1, S2 and S3 diets consumed 1222.38±39.18, 1137.71±55.17 and 1136.11±45.41 g feed and 1075.70±34.41, 1015.81±49.26 and 1014.40±40.55 g of dry matter on average, respectively. During trial period, feeding of ewes with sorghum stover based diets in block form had no effect (P>0.05) on feed and DM intake. Similarly, dietary inclusion of guar meal did not show any effect (P>0.05) on feed and DM intake in ewes.The chemical composition of experimental diets used in the study is presented in th fortnight was 29.12±1.01, 27.45±1.33 and 27.42±1.10 kg, respectively. Statistically, there was no significant difference in fort night body weight changes (kg) in ewes fed S1, S2 and S3 diets from first to 10th fortnight.The initial body weights of the ewes fed S1, S2 and S3 diets were 23.34±0.99, 23.60±0.74 and 23.10±0.26 kg, respectively  and the th and 90th day of pregnancy and at 15 d before expected day of lambing in ewes fed S1, S2 and S3 diets are presented in th day of pregnancy, lowest (P<0.01) on 15th day of pregnancy and intermediary on 15 d before expected day of lambing. However, there was no significant difference among the ewes fed different experimental diets. No interaction was observed between the diet and period in lipid peroxidase concentration.The lipid peroxidation (nanomol/ml) and glutathione peroxidase (U/dl) concentrations assessed on 15th day of pregnancy, lowest on 15 d before expected day of lambing and intermediary on 15th day of pregnancy (Glutathione peroxidase concentration was significantly (P<0.001) highest on 90regnancy . Irrespeth day of pregnancy. Irrespective of days of blood collection, the overall progesterone concentration (P> 0.05) was comparable among the three diets (The progesterone (ng/ml) levels irrespective of diets was higher (P<0.001) at 15 d before expected date of lambing, while the concentration was lower on day 15 and intermediate on 90ee diets .The onset of estrus after vaginal sponge removal was recorded as 26.32, 26.43 and 28.23 hours in ewes fed S1, S2 and S3 diets, respectively and the complete block feeding and replacement of 50% of groundnut cake -nitrogen with guar meal did not influence the time taken for the animals to exhibit estrus after the sponge removal.The per cent non-return rate and conception rate were 85.71, 100 and 85.71 in ewes fed S1, S2 and S3 diets. There was no significant (P>0.05) difference among three groups.The number of matings per conception did not differ significantly in ewes fed S1, S2 and S3 diets and was 1.00 for all experimental groups. The lambing rate was expressed as number of lambs born per number of ewes mated and was 100% in ewes fed S2 diet and in S1 and S3 diets was 85.71%. The per cent of lambs born to total number of ewes in group was 85.71, 100, and 85.71 in ewes fed S1, S2 and S3 diets, respectively. The lambing rate and per cent of lambs born did not differ significantly (P>0.05) in ewes fed S1, S2 and S3 diets.The average litter size for all the three dietary groups was 1.0 and did not differ between three groups. The duration of gestation did not seem to be influenced by the dietary variation and was recorded as 148.33±1.87, 150.33±1.34 and 153.33±2.89 days in ewes fed S1, S2 and S3 diets, respectively.th day and 5.08±0.22, 4.41±0.28 and 4.39±0.23 on 30th day in S1, S2 and S3 groups, respectively (th and 30th day.The body weights (kg) of lambs were 2.23±0.13, 2.21±0.13 and 2.24±0.14 at birth and 3.56±0.23, 3.14±0.19 and 3.25± 0.24 on 15ectively . The feeet al. (2016) [et al. (2014) [et al. (2010) [The results of the present study showed that inclusion of GM by replacing of 50% GNC nitrogen and feeding of complete diet in block form had no effect (P>0.05) on average DMI . The lac. (2016)  who repo. (2014) . Similar. (2010)  in cross. (2010)  in sheepth fortnight was due to usual gain in weight in pregnant animals because of foetal growth. The inclusion of guar meal had no adverse effect (P>0.05) on fortnightly body weights in ewes and similar body weights were observed in all three treatments without any significant difference. Similar to current findings, no effect (P>0.05) on live weight was observed with corn gluten meal (CGM) in beef cows [et al. (2015) [Nigella Sativa meal (NSM), which might be due to the presence of high unsaturated, essential fatty acids and amino acids in NSM. While, feeding of ewes with CGM as substitute to SBM decreased (P<0.05) body weights were observed in the studies of Liamadis and Milis (2007) [No loss in BW of ewes was observed during the trial period and there was a gradual increase in BW in all groups throughout the experimental period . The graeef cows  or driedeef cows . On cont. (2015)  in ewes s (2007) , furtheret al. (2019) [et al. (2012) [In this study, there was also no significant (P>0.05) difference in BW changes in ewes fed with block (S2) or conventional (S1) diet. Similar results were reported by Ibrahim (2008)  who repo. (2019)  and Laza. (2012) .th day, 90th day of pregnancy and 15 days before expected day of lambing  among the three dietary groups on 15 lambing . Higher s period . The raps period , 29. Mors period . Highestregnancy , 32. Theth day of pregnancy and 15 days before expected day of lambing. This indicated a greater stress during these periods. Similar results were reported by Weber and Kerr (2011) [et al. (2012) [et al. (2016) [Irrespective of dietary variation, lipid peroxidation and antioxidant enzyme activities varied across the time periods of blood collection. The lipid peroxidation was higher at 90r (2011)  who obse. (2012)  also rep. (2016)  observedNigella Sativa meal.The progesterone concentration increased (P<0.001) with the progress of pregnancy and did not differ among the dietary treatments (P>0.05) during all the 3 blood collections . IncreasThe reproductive traits of Deccani ewes are presented in et al. (2018) [et al. (2015) [et al. (2015) [Nigella Sativa meal in ewes.Many authors reported no effect on feeding various unconventional protein supplements on reproduction performance in animals. Erdogan . (2018)  observed. (2015)  also obs. (2015)  also obset al. (2017) [In the current study, densification of complete diet did not show any significant effect on conception rate and lambing rate. Similar findings were also reported by Salman . (2017)  and Ibra. (2017)  who obseet al. (2014) [et al. (2018) [et al. (2015) [et al. (2015) [Nigella Sativa meal compared to control group. The higher birth weight of lambs born to ewes fed on Nigella Sativa meal might be related to that protein requirements were adequate and rapidly degradable.Inclusion of guar meal and physical form of feeding did not show any effect (P>0.05) on body weights of lambs at birth, on day 15 and on day 30 . Similar. (2014)  observed. (2018)  also rep. (2015)  found th. (2015)  recordedIn the present study, physical form of the diet did not show any significant (P>0.05) effect on lamb birth weights. Similar findings were reported by Ibrahim (2008)  who obseThe conventionally used groundnut cake could be satisfactorily replaced by guar meal up to 50% on nitrogen basis in sorghum stover based complete feed blocks without any adverse effect on antioxidant status, progesterone concentration, conception rate, non return rate, no of matings per service, lambing rate, body weights of lambs at birth, 15 and 30 days of age. Further it was observed that densification of complete diets had no effect (P>0.05) on the conception rate and lambing rate in ewes.S1 Data(XLSX)Click here for additional data file.


The tumor microenvironment is crucial in tumourigenesis, response to therapy, and elimination of tumor cells. Tumor-infiltrating lymphocytes (TILs) promote the host immune response and are associated with a better prognosis in colorectal cancer (CRC). This multicentric retrospective study evaluated the relationship between the presence and intensity of TILs and survival outcomes. A total of 651 patients from four Portuguese oncological centers who underwent surgical resection for stages II or III colorectal adenocarcinoma between 2016 and 2019 were included in this study. The mean age of the study population was 70 years; 58.2% were males. The median overall survival was 58.03 ± 1.29 months  55.50 - 60.56), and the median disease-free survival (DFS) was 53.02 ± 1.39 months (95% CI 50.29 - 55.74). Patients with high infiltrate  had significantly longer DFS i.e., 58.48 ± 1.84 months (95% CI 54.87 - 62.09 months) vs 49.22 ± 1.75 months (95% CI 45.79 - 52.64 months) in the group with absent or minimal infiltrate; p = 0.003. Assessing the side of the tumor, high infiltrate was associated with higher DFS (59.86 ± 2.36 months (95% CI 55.23 - 64.50 months) vs 49.60 ± 2.40 months (95% CI 44.90 - 54.29 months), p = 0.011). This work reinforces the importance of research into possible prognostic and predictive factors in patients with CRC. Colorectal cancer (CRC) is the fourth most common malignant disease and the third leading cause of cancer deaths worldwide. In 2020, there were an estimated 1.9 million newly diagnosed CRC patients and 935,000 CRC-related deaths worldwide [There are well-known clinical and histological prognostic factors for CRC. However, it is increasingly clear that the tumor microenvironment (TME) plays a role in the prognosis and response to therapy in solid tumors . The TMEIn this study, the authors retrospectively evaluated data on CRC patients to assess the relationship between the presence and intensity of TILs and survival outcomes.This multicentric retrospective study included 651 patients from four Portuguese oncological centers . All patients were >18 years of age and underwent surgical resection for stage II or III colorectal adenocarcinoma during the calendar years 2016 to 2019.Exclusion criteria included patients who underwent biopsy or endoluminal treatment only and an absence of detailed information in histopathologic reports. When multiple tumors were present, the highest-stage tumor was assessed. Further data on histological diagnosis, histological grade, pathological tumor-node-metastasis (TNM) stage, and tumor location  were obtained from the reports. The TIL grade score  was also retrieved from the pathological report ,6. When Statistical analysis involved descriptive statistics (absolute and relative frequencies) and inferential statistics. Fisher’s test, logistic regression, Cox regression, and Kaplan-Meier survival analysis were used. The level of significance to reject the null hypothesis was set at (α) ≤ 0.05. Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS) version 28.0 . Overall survival (OS) was defined as the time from diagnosis to death due to any cause, and disease-free survival (DFS) was defined as the time from diagnosis to the first of either disease recurrence or death . The survival analyses were performed using the presence of high or low/absent TILs scores. This study was approved by the Ethical Committee for Health of Hospital Professor Doutor Fernando Fonseca .The demographic characteristics of the study population are described in Table Not all pathology reports described the inflammatory infiltrate with the terms “peritumoral infiltrate” and “intratumoral infiltrate”. A total of 16 missing values for "peritumoral infiltrate" and 229 for "intratumoral infiltrate" were detected. A total of 259 patients (39.8%) were classified as high infiltrate .Survival analysesThe median OS was 58.03 ± 1.29 months  55.50 - 60.56) and the median disease-free survival (DFS) was 53.02 ± 1.39 months (95% CI 50.29 - 55.74).The presence of high infiltrate was associated with better survival outcomes. There was a significant association between the presence of high infiltrate and DFS. The mean DFS in the high infiltrate group was 58.48 ± 1.84 (95% CI 54.87 - 62.09), compared with 49.22 ± 1.75 (95% CI 45.79 - 52.64) in the group with absent/minimal infiltrate (p = 0.003) , a statistically significant difference was not found (p = 0.432).Assessing the right and left colon cancer patients, we found that the presence of high infiltrate was associated with better survival outcomes in left, but not in right colon cancer. In this group of patients, the mean DFS in the high infiltrate group was 59.86 ± 2.36 months (95% CI 55.23 - 64.50 months), compared with 49.60 ± 2.40 months (95% CI 44.90 - 54.29 months) in the group with absent or minimal inflammatory infiltrate (p = 0.011)  compared with the absent or minimal inflammatory infiltrate (51.02 ± 1.90 (95% CI 47.30 - 54.75); p = 0.037). In stage III patients, although the DFS in the high infiltrate group was superior compared with the absent/minimal infiltrate group (52.16 vs 45.01 months), a statistically significant difference was not found (p = 0.106). Regarding OS evaluation, no significant differences were found in either stage. In addition, in univariate analysis, the staging and presence of perineural invasion were significantly associated with DFS, but not with OS.Most CRC diagnoses occur at early stages, allowing for better survival. However, after resection with curative intent, approximately 20% of patients with stage II or III CRC will have a disease recurrence. Certain well-documented clinical and histological risk factors are associated with a higher probability of recurrence. In stage III patients and those in stage II with risk factors, adjuvant treatment is recommended to reduce the recurrence risk .Our study describes the clinical and histological characteristics of stage II and III CRC patients who underwent surgical resection in four Portuguese institutions over four years. As expected, initial tumor staging was identified as a prognostic factor in our sample, with stage III being associated with worse DFS compared to stage II. The OS was not significantly different among stage III patients, likely due to the short follow-up time.Since the 1970s, evaluating the immune environment around the tumor has been described as an essential factor in CRC prognosis. The immune response is an important factor in tumor behavior and affects tumor development and growth. Infiltration of immune cells such as lymphocytes, macrophages, and dendritic cells predicts the immune response, promoting tumor elimination ,9. The lThe lymphocyte subpopulations in the TME in CRC allowed the establishment of response patterns. A multiplex immunohistochemical method identifies specific immune cells according to the surface markers. Cells such as regulatory T cells and M2 tumor-associated macrophages play mostly tumor-promoting functions. On the other hand, macrophages of M1 phenotypes or T cells CD8+ are related to antitumor activity, driving a better prognosis . SeveralIn our study, the lymphocytic infiltrates were evaluated according to anatomopathological reports previously made. This limits the uniformity of the evaluation as it is subject to inter-observer variation. Despite these limitations, the benefit of the presence of inflammatory infiltrates in TME is notable, highlighting the importance of routinely reporting this variable for colon cancer surgical specimens in clinical practice. The increase in DFS was almost 10 months in the patients with high infiltrate compared to those with absent/minimal infiltrate.In most recent studies, the presence of MSI has been reported as a prognostic factor in CRC patients. In addition, MSI-H is associated with a higher response rate with immune checkpoint inhibitors ,16,20. TThe tumor side is a known prognostic factor given the differences in the biological origin of the tumors. The tumor location also has therapeutic implications, mostly related to anti-epidermal growth factor receptor antibodies . In our Our study confirms the prognostic importance of inflammatory infiltrates in CRC tumors, with significant differences observed in DFS. The study's limitations include the fact that it is a retrospective analysis that depends on previous pathological reports. In future studies, it would be valuable to include pathological re-analysis  as well as an increased sample size involving the participation of other centers.This work reinforces the importance of research into possible prognostic and predictive factors in patients with CRC. Identifying factors that alter prognosis has implications for the type of surveillance to be performed and for the possibility of adjuvant treatment. The study of the TME is a critical factor in understanding oncological treatment. The development of methods that evaluate components of the immune system in the tumor stroma allows the relationship between their presence and responses to therapies and survival outcomes to be assessed.In our study, the presence of TILs was significantly associated with DFS. The results obtained are concordant with what has been previously reported in individual studies and meta-analyses. However, there is a need for further research on this topic, including new markers that are easily accessible in clinical practice.


They also had a higher rate of bone marrow blast < 5% (p = 0.004), a higher proportion of low and int-1 risk according to IPSS score (p = 0.023), and lower median platelet (PLT) count (p < 0.001). To date, in the del20q cohort, 21 patients (30.4%) received no treatment, 42 (61.0%) were treated with erythropoiesis-stimulating agents (ESA), 3 (4.3%) with hypomethylating agents, and 3 (4.3%) with other treatments. Among 34 patients evaluable for response to ESA, 21 (61.7%) achieved stable erythroid response according to IWG 2006 criteria and 13 (38.2%) were resistant. Nine patients (13.0%) progressed to acute myeloid leukaemia (AML) after a median time from diagnosis of 28 months (IR 4.1–51.7). The median overall survival (OS) of the entire cohort was 60.6 months (95% CI 54.7–66.4). the 5-year cumulative OS was 55.9% (95% CI 40.6–71.2). Conclusion: According to our results, we hypothesize that MDSs with isolated del 20q may represent a distinct biological entity, with peculiar clinical and prognostic features. The physio-pathological mechanisms underlying the deletion of the chromosome 20 long arm are still unclear and warrant future molecular analysis.Aims: To define the peculiar features of patients with the deletion of the chromosome 20 long arm (del20q), data from 69 patients with myelodysplastic syndromes (MDSs) and isolated del20q, followed by the Gruppo Romano-Laziale Sindromi Mielodisplastiche (GROM-L) and Ospedale Torrette of Ancona, were collected and compared with those of 502 MDS patients with normal karyotype (NK-MDS). Results: Compared to the NK-MDS group, patients with del20q at diagnosis were older ( Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid disorders characterized by defective bone marrow haematopoiesis that cause peripheral cytopenias, recurrent cytogenetic abnormalities, and a variable risk of progression to acute myeloid leukaemia (AML) .Given the heterogeneity of the disease and the different clinical behavior in terms of leukemic evolution, there have been many efforts to identify patients with similar biological, clinical, and prognostic features . These eDeletion of the long arm of chromosome 20 (del20q) has been observed in a small subset of MDS patients, both alone and with other cytogenetic abnormalities. In particular, del20q was reported in only 2% of the cohort for the validation of the IPSS and in 1.3% of the MDS included in the Groupe Francophone des Myélodysplasies (GFM) registry . DescripWe collected data from all patients with isolated del20q MDSs treated in 12 hematologic centers to define their peculiar clinical and prognostic features in comparison to MDS patients of same prognostic group (IPSS) with normal karyotype (NK-MDS). The aim of this data collection is to evaluate the role of this karyotypic abnormality as the hallmark of a new possible subset in the MDS landscape.We retrospectively collected data of all consecutive patients diagnosed as having MDS with isolated del20q between January 2002 and December 2016 in 12 haematologic centres of the Gruppo Romano-Laziale Sindromi Mielodisplastiche (GROM-L) and Ospedale Torrette of Ancona .For enrolment in this study, the required criteria were a confirmed diagnosis of MDS according to WHO 2016 classification and the presence of isolated deletion of the long arm of chromosome 20. The presence of inherited bone marrow failure syndromes with del20q was an exclusion criterion.Complete peripheral blood and bone marrow (BM) features were recorded for any eligible patients, together with follow-up data and the date of leukemic evolution. The International Prognostic Scoring System (IPSS) was used for the prognostic stratification of all patients. Characteristics of patients with isolated del20q were compared with those of 502 NK-MDS patients included in the GROM-L registry between January 2002 and December 2016.Chromosome banding analysis was performed after 24 h on an unstimulated culture of BM cells using standard techniques. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature criteria (2016). Cytogenetic analysis was performed at local specialized laboratories in each participating center.A descriptive analysis of the demographic and clinical characteristics of the patients was performed, including the median and interquartile range (IQR) for continuous variables, and the absolute and relative frequencies of categorical variables.Overall survival (OS) was defined as the time from diagnosis to death from any cause or to the date of the last follow-up. Cumulative incidence of AML evolution was defined as the time from diagnosis to either the evolution into AML or the last follow-up.Survival curves were estimated according to the Kaplan–Meier product limit method and were tested for significant differences using the log-rank test in univariate analysis and by means of the Cox regression model in multivariate analysis.p ≤ 0.05 as statistically significant. All analyses were performed using the IBM SPSS statistics software.In all analyses, 95% confidence intervals (95% CI) were reported for the main summary statistics and all statistical comparisons were based on two-tailed tests accepting The study population included 69 patients with isolated del20q from a total number of 876 MDS patients with available karyotype, with a global incidence of 7.8%. The main clinical features of these patients are reported in p = 0.020) and mainly male (p = 0.006). They also had a higher rate of marrow blast count <5% (p = 0.004), a higher proportion of low/intermediate-1 risk according to IPSS (p = 0.023), and a lower median PLT count at diagnosis (p < 0.001). Limiting the comparison to patients with a blast count of <5%, patients with isolated del20q continued to have a lower median PLT count at diagnosis than the NK-MDS population [30/58 (51.7%) vs. 87/290 (30%) PLT count < 100 × 109/L (p = 0.001)].Compared with NK-MDS, patients with isolated del20q were older (p = 0.920), hemoglobin (Hb) (p = 0.849), and ferritin levels (p = 0.741).No significant differences were found between the two groups regarding leukocytes (WBC) count  had a PLT count <100 × 10At the last available follow-up, after a median time from diagnosis of 12.4 months (IQR 4.4–35.9), a watch-and-wait strategy was performed in 21 patients (30.4%), without starting any specific treatment.Among the remaining 48 patients, 42  were treated with erythropoiesis stimulating agents (ESA), 3 (4.3%) with hypomethylating agents, and 3 (4.3%) with other treatments .Treatment with ESA started after a median time from diagnosis of 5.8 months (IQR 1.2–26.4): 27 patients (64.2%) received EPOα, 13 (31.0%) EPO-β, 1 (2.4%) darbopoietin, and 1 (2.4%) EPO-ζ. Median endogenous EPO levels at baseline of ESA treatment were 43.0 mU/L (IQR 20.7–50.6). Overall, 34 patients were fully evaluable for the response in accordance with the IWG 2006 criteria. Of these, 21 (61.8%) achieved a stable erythroid response , after a median time from ESA start of 3.2 months (IQR 2.1–5.6), while 13 (38.2%) were resistant. The median duration of response to ESA was 14.0 months (IQR 4.8–33.7).Nine patients (13.0%) progressed to AML, with a median time to evolution of 28 months (IQR 4.1–51.7); 3-year and 5-year cumulative incidences were 10.2% (95% CI 1.6–18.8) and 20.0% (95% CI 4.9–35.1), respectively .At the last follow-up, 30 patients (43.5%) died (13 for MDS/AML-related causes and 17 for unrelated causes), 14 (20.3%) were lost to follow-up, and 25 (36.2%) were alive.The 3-year and 5-year OS of patients with isolated del20q were 62.9% (95% CI 49.2–76.6) and 55.9% (95% CI 40.6–71.2), respectively, with a median OS of 60.6 months (95% CI 54.7–66.4) .9/L vs. ≥100 × 109/L), WBC (<2.5 × 109/L vs. ≥2.5 × 109/L), marrow blasts (<5% vs. ≥5%), IPSS (low vs. intermediate-1), and transfusion need (no vs. yes).The following features at diagnosis were evaluated as risk factors for cumulative incidence of AML evolution and OS in patients with isolated del20q: age (<75 years vs. ≥75 years), gender, Hb (<10 g/dL vs. ≥10 g/dL), PLT (<100 × 10p < 0.001)  for patients with ≥5% vs. 2.6% blasts (95% CI 0–7.5) and for patients with <5% blasts (< 0.001) . All othp < 0.001), age <75 years at diagnosis (p = 0.008) and Hb values >10 g/dL at diagnosis (p = 0.050); patients with age <75 years had a 3-year OS of 72.0% (95% CI 53.9–90.1) vs. 53.1% (95% CI 32.7–73.5) in patients aged ≥75 years. The remaining factors were not statistically significant.At univariate analysis for OS, factors associated with better survival were low IPSS  (p = 0.021) (Comparing the cumulative incidence of AML evolution in patients with isolated del(20q) in the NK-MDS group, there was no statistically significant difference (= 0.973) . On the = 0.021) .We evaluated the clinical characteristics of 69 patients, which represent 3% of all MDS collected in the GROM-L registry from 2002 to 2016. As far as we know, this cohort of MDSs with isolated del20q is one of the widest in the current literature with the specific aim to compare this subtype with other MDSs; thus, we analyzed our results referring to the similar report of the GFM in 2011.In the GFM analysis, patients with isolated del20q significantly differed from other MDS by higher reticulocyte count, lower platelet count, and lower mean percentage of marrow blasts . These l9/L in more than 50% of our patients, in accordance with GFM data. As a matter of fact, thrombocytopenia is highlighted as the commonest clinical distinctive feature in MDSs with del20q up to now.However, the most significant feature at diagnosis was a reduced PLT count, with values <100 × 10As already reported by the GFM study, thrombocytopenia was associated with Hb values >12 g/dL in 13% of the subjects in our cohort, and in 7% of cases it represents the only cytopenia at diagnosis; these patients were all males and with marrow blasts <5%, thus raising the need of a careful differential diagnosis with idiopathic thrombocytopenic purpura (ITP). This point is strengthened by two different observations; first, it has already been pointed out in the literature that del20q is the most frequently encountered cytogenetic anomaly in patients with isolated thrombocytopenia at onset . All theIt is important to underline that the presence of del20q, as well as -Y and +8, is not considered to be MDS-defining in the absence of diagnostic morphologic features of MDSs; morphologic examination remains crucial in the differential diagnosis between ITP and MDS. It is worthy of note, to this respect, that some specific morphological features (hypogranulated and vacuolized neutrophils and neutrophil erythrophagocytosis) have already been reported in MDS subjects with del20q .Moreover, patients with del20q were significantly older, with a median of 76 years at diagnosis, and were prevalently males compared to NK-MDS. These features were not reported in the GFM analysis, thus requesting further confirmations in larger series.Regarding the treatment of patients with isolated del20q, about one third of them did not need any therapy and were managed with clinical monitoring only, while the vast majority received ESA, especially EPO-α. Erythroid response, according to the IWG criteria in these patients, was in line with other MDS subtypes, with more than 60% presenting a stable and durable response; thus, our data confirm the use of recombinant erythropoietin as the appropriate first therapeutic choice in these subjects.The relatively good prognosis of patients with isolated del20q is also indicated by the low rate of AML evolution, similar to that reported in NK-MDS, and by the median OS of >60 months.9/L) was not significant.According to GFM data, the OS of these patients was influenced by anemia and thrombocytopenia. In our cohort, the presence of Hb < 10 g/dL was associated with a lower OS in both univariate and multivariate analysis; on the contrary, PLT count at diagnosis . This observation may in part be explained by the significantly older age of patients with isolated del20q, but could also have a biological basis.Although the presence of isolated del20q is associated with a relatively favourable prognosis compared to patients with other karyotypic alterations, the OS remains worse compared to NK-MDS (Large cohort studies have led to the identification of a putative commonly deleted region (CDR), located in the proximal area adjacent to the centromere 20q11.1–11.21) near the MAFB gene and in the sub-telomeric region 20q13.33) near the ADNP gene . While t.1–11.21 .33 near We are aware that the present retrospective evaluation has some important limitations. First of all, MDS diagnosis was not centrally revised, with possible discrepancies in the evaluation of dysplastic marrow features. Moreover, the vast majority of our patients were diagnosed many years ago, when mutational analyses were not performed. In addition, NGS techniques are still not used in the routine clinical activity. As a consequence, we do not have any mutational data to report in the present study, but we are planning a prospective phase to clarify this very important issue. Finally, isolated del20q has been reported also in other hematological disorders, both inherited and acquired, such as myeloproliferative neoplasms (MPN). Even if the presence of dysplastic marrow features and the absence of other signs typically reported in MPN  allowed us to discriminate our cases from MPN, we cannot exclude misdiagnosis at all.In conclusion, MDSs with isolated del20q constitute a small but quite homogeneous subtype from a clinical and prognostic point of view, with a biological substrate likely different from other MDS. Differential diagnosis with ITP could be difficult in elderly patients with isolated thrombocytopenia, and cytogenetic or FISH analysis should be considered in some cases of refractory ITP.


To evaluate the concordance of the risk of neonatal respiratory morbidity (NRM) assessed by quantitative ultrasound lung texture analysis (QuantusFLM) between twin fetuses of the same pregnancy. Prospective study conducted in twin pregnancies. Fetal ultrasound lung images were obtained at 26.0–38.6 weeks of gestation. Categorical (high or low) and continuous results of the risk of NRM were compared between twins. Fetal ultrasound lung images from 131 pairs (262 images) of twins were included. The images were classified into three gestational age ranges: Group 1 ; Group 2  and Group 3 . Concordance was good in Groups 1 and 3 and moderate in Group 2. In Groups 2 and 3 at least one fetus presented high-risk results in 26.5% and 11.6% of twin pairs, respectively. Only gestational age < 32 weeks, gestational diabetes mellitus, and spontaneous conception were associated with a high risk of NRM in Group 2. There was good concordance of the risk of NRM between twins < 30.0 weeks and > 34.0 weeks. From 30.0 to 33.6 weeks 26.5% of the twin pairs had discordant results, with moderate concordance of the risk of NRM. There is always clinical concern that fetal lung maturity (FLM) may differ between twins. Therefore, prediction of FLM may be helpful when premature and early term delivery of twins is expected.Discordant neonatal respiratory morbidity (NRM), such as respiratory distress syndrome (RDS) has been described in twin pregnancies3. The rate of late preterm delivery may be as high as 50%4. The development of RDS and transient tachypnea of the newborn (TTN) are major causes of neonatal morbidity and have been described by many authors7, with several studies having evaluated the most reliable test to predict these outcomes11. Tests to estimate FLM by measuring surfactant in amniotic fluid were developed in the 1960s and 1970s after advances in the understanding of fetal lung development. However, these tests required an invasive procedure to extract and later analyze the amniotic fluid, and despite their excellent performance in determining FLM, their use has practically disappeared12. Indeed, an invasive procedure was a main limitation for studies in twin pregnancies using lamellar bodies, TDx-FLM, lecithin/sphingomyelin ratio or other similar tests. Therefore, information on fetal lung maturity concordance along pregnancy between co-twins is scarce, due to the invasive nature of amniocentesis, and has not been extensively studied.The rate of pregnancy complications is higher in twin pregnancies resulting in increased neonatal morbidity linked to prematurity17.In the search for a non-invasive method to obtain FLM information carried out in obstetric ultrasound in the last decade, approaches based on quantitative ultrasound analysis have specifically been explored in fetal medicine to determine the potential of ultrasound to predict FLM19, included a significant number of high-risk pregnancies, but the performance of this tool in particular groups of patients such as twin pregnancies has not been specifically addressed. The clinical decision to deliver twin pregnancies preterm is challenged by the presence of more than one fetus with potentially different lung maturity status.Previous studies using quantitative ultrasound lung texture analysis (QuantusFLM)The aim of the study was to evaluate the concordance of the risk of NRM assessed by QuantusFLM between twin fetuses of the same pregnancy at different gestational age ranges.19. Briefly, a lateral axial section of the fetal thorax at the level of the 4-chamber cardiac view without any type of postprocessing manipulation was acquired, collected, and stored in the original Digital Imaging and Communication in Medicine format. The images were anonymized by removing patient information and labeled with new identification numbers and the gestational age at acquisition and later classified into three gestational age ranges: Group 1 (26.0–29.6 weeks), Group 2 (30.0–33.6 weeks) and Group 3 (34.0–38.6 weeks). The analysis included only cases in which the images of both fetuses were available in the appropriate gestational age group.This prospective study was performed at the Maternal–Fetal Medicine Department at the Hospital Clinic of Barcelona in collaboration with the Hospital Clínico Universidad de Chile (Chile) and Hospital Del Valle in Cali (Colombia). Eligible cases included consecutive cases of twin pregnancies between 26.0 and 38.6 weeks of gestation undergoing follow-up ultrasound during routine prenatal visits from December 2017 to March 2020. Ultrasound images were obtained following an acquisition protocol described elsewherewww.quantusflm.com  and analyzed using the new QuantusFLM version 3.020. This software automatically delineates a region of interest in the fetal lung and calculates an NRM risk score (defined as the occurrence of either RDS or TTN) as a continuous variable. To evaluate the risk of NRM, continuous output NRM risk scores were binarized using the optimal cut-off point threshold computed as that maximizing accuracy in the test images, thereby obtaining a categorical result . The results were considered concordant when both twins were low-risk or high-risk of NRM and discordant when the risk of one twin differed from the co-twin.Images fulfilling the quality criteria were loaded via the Internet by engineers at the coordinating center through restricted access to the commercial software websiteAll patients included in the study provided written informed consent for the use of ultrasound images and perinatal data. The study was performed in accordance with relevant guidelines and regulations and approved by the Institutional Review Board of the Hospital Clinic of Barcelona (HCB/2017/0642).2 and Fisher exact tests. Concordance was assessed with the McNemar test in paired nominal data and intraclass correlation coefficient (ICC) with two-way mixed-effects model using absolute agreement in continuous data. We considered ICC values < 0.5, between 0.5 and 0.75, between 0.75 and 0.9, and > 0.90 as poor, moderate, good, and excellent concordance, respectively21.Quantitative variables were assessed using the Shapiro–Wilk test for normality, and normally distributed variables were compared using the t-test and expressed as mean and standard deviation/standard error of the mean (SD/SEM). Non-normally distributed quantitative variables were compared using the Mann–Whitney U test and expressed as median and interquartile range (IQR: p25–75). Qualitative variables were compared using the XUnivariate logistic regression analysis was performed in each group to explore variables associated with high risk of NRM. A p-value < 0.05 was considered significant. Data were analyzed using STATA, v.15.0 .A total of 306 images were obtained; 32 10.4%) were non-eligible and discarded after image quality control or having been stored in incorrect formats, and 12 (3.9%) were excluded after imaging processing due to the impossibility of obtaining an image in one of the twins to compare. The study included 131 pairs of twins (262 images). The flowchart of the eligible images is depicted in Fig. 0.4% wereEach pair of twins contributed once and the images were classified and analyzed in three gestational age ranges: Group 1 ; Group 2  and Group 3 .The baseline and clinical characteristics of the 131 women are shown in Table Figure Group 1 (26.0–29.6 weeks) showed high risk results in all but 1 case (2.6%) achieving global concordance of 97.4% (38/39). Group 2 (30.0–33.6 weeks) presented global concordance in 73.5% (36/49) of cases. with 7 of the 36 pairs with concordant results showing high-risk results for NRM in both fetuses. Of the 13 pairs with discordant results, 61.5% (8/13) of high-risk results corresponded to the second twin. Therefore, in 20/49 (40.8%) twin pairs in Group 2, one or both fetuses had high-risk results for NRM. In group 3 (34.0–38.6 weeks) the global concordance was 88.4%; in 5/43 (11.6%) twin pairs, one fetus presented high-risk results for NRM with no cases of both fetuses scoring high-risk for NRM. In Group 1, the discordant pair of twins was dichorionic. In Group 2, 18/49 (36.7%) twin pairs were monochorionic and 31/49 (63.3%) were dichorionic. The percentage of concordant results in monochorionic (72.2% [13/18]) versus dichorionic (74.2% [23/31]) twins, was not statistically significant (p = 0.880). The discordant pairs in this group were 27.8% (5/18) monochorionic and 25.8% (8/31) dichorionic. In Group 3, 10/43 (23.3%) twin pairs were monochorionic and 33/43 (76.7%) were dichorionic. The percentage of concordant results in monochorionic (90.0% [9/10]) versus dichorionic (87.9% [29/33]) twins, was not statistically significant (p = 0.855). The discordant pairs were 10.0% (1/10) monochorionic and 12.1% (4/33) dichorionic.The analysis for continuous QuantusFLM results showed good concordance in Groups 1 and 3, [ICC = 0.781 ] and [ICC = 0.900 ], respectively; while concordance was moderate in Group 2 [ICC = 0.522 ].The mean gestational age at fetal lung ultrasound examination was lower for high-risk vs. low-risk results in all groups; Group 1 (27.5 vs. 29.4 weeks); Group 2 (31.2 vs. 32.0 weeks) and Group 3 (34.8 vs. 35.4 weeks), being statistically significant only in Group 2 (p ≤ 0.001).Other variables were evaluated to determine the characteristics potentially associated with high-risk NRM results. Figure Table We prospectively evaluated for the first time the concordance of the risk of NRM between twins with non-invasive quantitative ultrasound lung texture analysis, obtaining 97.4% (Group 1), 73.5% (Group 2) and 88.4% (Group 3) concordance in the risk of NRM. Indeed, the risk of NRM was discordant between twin pairs in 2.5% (1/39), 26.5% (13/49) and 11.6% (5/43) in Groups 1, 2 and 3, respectively. We found 97.4% (77/78) of fetuses with high-risk results in Group 1, 27.6% (27/98) in Group 2 and 5.8% (5/86) in Group 3. These results achieved good concordance in Groups 1 and 3, being moderate in Group 2. Knowing the status of fetal lung maturity may be helpful in clinical practice since frequent obstetric complications impel delivery planning in twin pregnancies in the late-preterm and early-term weeks of gestation. In such circumstances delaying delivery could be ambiguous, and information of lung maturity may help to weigh maternal and fetal risks. Our univariate logistic regression analysis showed that spontaneous conception, gestational diabetes mellitus and gestational age < 32 weeks were associated with a high-risk of NRM in Group 2. Variables associated with high-risk results were not found in Groups 1 or 3.A decade ago, the prediction of fetal lung maturity relied almost solely on laboratory tests of amniotic fluid obtained invasively (amniocentesis). Previous studies using these tests have described similar differences in FLM to those of our study at a given gestational age and many gestational factors had been proposed as predictors of FLM concordance.22. In a study including 42 pairs of twins, Leveno et al. reported a high correlation between lecithin/sphingomyelin (L/S) ratios between twins A and B which was not related to birth order, infant sex, birthweight discordance, or zygosity23. Following amniocentesis in 47 twin pregnancies (26–36 weeks), Winn et.al, reported that FLM was biochemically comparable between twins A and B24, being in line with our results showing concordant results in 97.4%, 73.5% and 88.4% of twins in Groups 1, 2 and 3, respectively. Similar to our results, McElrath, et al. found that after 31.0 weeks, twin gestations had significantly higher levels of TDx  of FLM values25. Another study by Whitworth et al. showed that when L/S ratios were stratified by gestational age at amniocentesis, the mean percentage difference of the ratios was 25% at ≤ 32 weeks and 16% at > 32 weeks. In all the sets of twins, in which the ratios were discordant, the gestational age at amniocentesis was ≤ 32 weeks26. Dobbie et al. explored the correlation of biochemical ratios  of FLM tests in 32 pairs of twins. They found a weak correlation for L/S ratio but a much-improved correlation for PG/S and PI/S. Concordance was greater in monochorionic pregnancies1. In a large study of 132 dichorionic and 125 monochorionic twins, Tsuda et al. examined the amniotic lamellar body count (LBC) and found that the concordance of the LBC results for the cut- off value was also significantly lower in dichorionic than in monochorionic twins27.Regarding gestational age and chorionicity, Mackenzie performed amniocentesis in 92 pairs of diamniotic twins and found that at extremes of gestational age , twins were 100% concordant in lung status, while lung concordance was significantly lower in twins of 33–35.9 weeks, and even lower at 30–32.9 weeks. The influence of chorionicity, estimated fetal weight discordance and fetal gender concordance were not predictive of lung maturity28 and Steen et al.29.We found no association of fetal gender with the QuantusFLM results, but we did not test the results with the outcome at birth. However, the presence of RDS at birth was more prevalent in male newborns, similar to the studies by Mulla et al.31 and FLM tests33. It has also been associated with mixed results such as more frequent admission to neonatal intensive care units and greater birthweight but not with greater respiratory morbidity in twin pregnancies35. In our study gestational diabetes was a statistically significant predictor of high-risk results in Group 2; however, the number of cases was limited. This can be expected in these weeks of transition from pulmonary immaturity to maturity, since this association was not found in Group 3.Maternal diabetes has commonly been reported as a factor related to FLM40. Literature correlating fetal lung maturity and ART is scant. Tsuda et al., evaluated the impact of fertility treatment on NRM in twin pregnancies using amniotic LBC and found no statistically significant associations between fertility treatments and the rates of RDS/TTN41. We found a statistically significant association of IVF treatment to low-risk results in Group 2. When the analysis was performed by gestational age, we found that images with high-risk results from fetuses conceived by IVF were obtained at a mean gestational age of 31.9 weeks, compared to high-risk images of spontaneously conceived fetuses that were obtained at a mean gestational age of 30.9 weeks. This finding may explain why IVF has been found to be a protective factor in this group.Assisted reproductive techniques have been associated with adverse perinatal outcomes in twin pregnanciesThe aim of the present study was not to test performance but rather to explore the concordance of the high- or low-risk of NRM in prospectively followed twin pairs. This study showed a relevant number of cases of fetal lung immaturity, at least in one of the fetuses, in the gestational age ranges of Groups 2 and 3, which should be considered if delivery is expected. While in Group 2, 59.2% of pairs were already at low-risk, 11.6% in Group 3 remained at high-risk, in line with studies in which FLM was evaluated by invasive techniques.23, Mackenzie22 and Winn et al.24, who included no more than 92 pairs of twins. However, some limitations are of note: respiratory morbidity was not clinically addressed immediately after the test. However, the study was designed to evaluate the concordance of the test in utero between twins, and this may help in the clinical decision-making process. Questions about a possible more advanced or delayed lung maturation process throughout pregnancy have been raised for years regarding fetuses with conditions such as growth restriction or maternal diabetes. However, information on this topic is scarce due to the limitation of the invasive nature of amniocentesis. The data obtained in this study show that discordant lung maturation could be expected in different ranges of gestational age, and this should be taken as a proof of concept.The main strength of this study is first, the fact that a non-invasive technique was used to evaluate the individualized risk for NRM for each twin; and secondly, our study included a larger number of pairs than the studies by Leveno et al.Indeed, a multicenter study demonstrated that QuantusFLM has an accuracy of 86.5%, like that of invasive techniques, although a further study to evaluate the performance of this technique in twins is forthcoming. This information will be decisive to assess whether the tool could be added to the management of twins in clinical protocols or guidelines. In the meantime, while the information shown in this study may not by itself be decisive when planning delivery, knowledge of the probability of discordance in a particular range of gestational age can help. Indeed, this could be relevant where a neonatal unit is not available.In conclusion, there was good concordance of the risk of NRM between twins < 30.0 weeks and > 34.0 weeks. From 30.0 to 33.6 weeks 26.5% of the twin pairs had discordant results. Gestational age < 32 weeks, gestational diabetes mellitus and spontaneous conception were found to be associated to high-risk results of NRM in this group. In 11.6% of pregnancies in the group above 34 weeks, at least one fetus of these pairs, showed high-risk of NRM. This information may be helpful to plan and guide obstetricians in the clinical decision-making process.Supplementary Figures.