Gastroduodenal artery (GDA) stump erosion hemorrhage is a fatal complication after pancreaticoduodenectomy. This study aimed to determine whether GDA stump wrapping with the teres hepatis ligament during pancreaticoduodenectomy decreased the incidence of postpancreatectomy hemorrhage (PPH).We reviewed 307 patients who had undergone pancreaticoduodenectomy between March 2019 and June 2022. The patients were divided into two groups according to application of GDA stump wrapping with the teres hepatis ligament: GDA wrapping group (165 patients) and no-wrapping group (142 patients). The perioperative data were compared between the groups.P = 0.029; GDA hemorrhage, 5.6% vs 0.6%, P = 0.014). No difference was observed in the incidence of clinically relevant postoperative pancreatic fistula, biliary leak, intra-abdominal abscess, delayed gastric emptying, 90-day mortality, and postoperative hospital stay between the two groups.The clinical characteristics were balanced between the two groups. Grades B and C PPH and GDA-stump-related hemorrhage were significantly reduced in the GDA wrapping group compared with the no-wrapping group (PPH B/C, 13.4% vs 6.1%, Wrapping GDA stump with the teres hepatis ligament reduced the incidence of GDA-stump-related PPH. Therefore, the wrapping technique is a simple and effective strategy to prevent PPH. Prospective studies are needed to confirm the benefit of this procedure. Pancreaticoduodenectomy (PD) is a standard surgical procedure for pancreatic head and periampullary carcinomas. This procedure is associated with high morbidity and mortality , 2 PostoSeveral methods have been developed to prevent POPF and GDA-stump-related PPH. Wrapping pancreatoenteric anastomosis or skeletal vessels with omental flaps or ligaments is one of the procedures to protect the surrounding organs against pancreatic juice –12. HoweBetween March 2019 and June 2022, the medical records of patients who underwent PD at the Second Affiliated Hospital of Zhejiang University and Affiliated Hospital of Jiaxing University were reviewed for eligibility. The exclusion criteria were no teres hepatis ligament available to create a wrapping or no creation of a pancreaticoenterostomy. Patients were divided into two groups depending on whether they had wrapping of the GDA stump by the teres hepatis ligament. It is each surgeon’s preference to or not to do the wrapping. All clinical, biochemical, and radiological data were retrospectively collected from the database. The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University (No: 2023–0667). All patients provided written informed consent before inclusion.Perioperative morbidity included PPH, POPF, biliary leak, delayed gastric emptying, wound infection, intra-abdominal abscess, pneumonia, and heart failure. PPH, POPF, and delayed gastric emptying were defined and graded according to the international consensus definitions of the International Study Group of Pancreatic Surgery (ISGPS) , 15, 16.Division of the GDA was routinely performed using 4–0 polypropylene sutures or two hem-o-lok clips. After the completion of PD, the teres hepatis ligament was mobilized after division of the falciform ligament close to the umbilicus. The teres hepatis ligament was separated from the liver parenchyma to ensure achievement of a ligament length of 10–15 cm. The blood supply and fat near the ligament were preserved. The prepared pedicled teres hepatis ligament was placed below the divided GDA stump and wrapped around it in a tension-free manner  and 165 with (GDA wrapping group) wrapping of GDA stumps (Table The perioperative characteristics are listed in Table P = 0.029). Eight patients (5.6%) developed GDA-stump-related PPH in the no-wrapping group, compared with only one patient (0.6%) in the GDA wrapping group (P = 0.014). The anatomical location of the main sites of bleeding in patients with clinically relevant PPH is listed in Table P = 0.557). Other complications included biliary leak, delayed gastric emptying, intra-abdominal abscess, pneumonia, and heart failure, and these did not differ significantly between the two groups. No differences were observed in 90-day reoperation and mortality between the two groups. The mean postoperative hospital stay was 28.0 ± 10.6 and 30.2 ± 17.3 days in the no-wrapping and GDA wrapping groups, respectively (P = 0.174).The postoperative complications are listed in Table  P = 0.01. The anaPPH remains one of the major complications after PD. However, it carries a high mortality of ~ 20% , 24. PPHThe wrapping technique using the omental flap and ligament was developed to protect the skeletonized vessels and pancreatic enteric anastomosis , 31–33. The wrapping technique was simple to perform either in open or minimally invasive surgery. The wrapping procedure to mobilize the teres hepatis ligament and wrap the GDA stump took an average of 5–10 min; thus, this step did not overly prolong operating time Table . The comP = 0.029) and GDA-stump-related hemorrhage , indicating the vessel-protective effect of wrapping. However, no difference in POPF was found between the two groups, indicating that the GDA stump wrapping did not lower the incidence of POPF. Wrapping had no obvious influence on biliary leak and delayed gastric emptying, and wrapping-associated complications reported in previous studies, such as intra-abdominal infection hepatic artery stenosis, did not increase in the wrapping group compared with the no-wrapping group. Despite the high mortality rate in cases of PPH, the lower rate of PPH in the wrapping group did not translate into a significantly lower mortality rate . This might have been due to the overall low rate of GDA-stump-related PPH. The application of interventional angiography and transcatheter arterial embolization for arterial hemorrhage also rescued most of the PPH. Eighty percent of the patients (4/5) in the wrapping group and 71.4% (10/14) in the no-wrapping group had successful hemostasis by transcatheter arterial embolization (data not shown). Thus, the mortality caused by PPH was too low to affect the statistical results.In the present study, we included a no-wrapping group as a control. The overall incidence of grade B/C PPH and clinically relevant POPF in the control group was 13.4% and 13.4%, respectively, which corresponded with previous studies in high-volume centers , 23. TheThis was a multicenter retrospective study with a small sample size, which may have led to selection bias. Therefore, well-designed randomized controlled trials are needed to verify the benefits of this technique in PD.In conclusion, our study showed that wrapping the GDA stump with the teres hepatis ligamentum decreased the incidence of GDA-stump-related hemorrhage and grade B/C PPH. Such a wrapping procedure, which is simple to perform in open or minimally invasive surgery, without increasing the operating time and other complications, can protect the GDA stump from pancreatic juice and prevent hemorrhage.


To ease water scarcity, dynamic programming, stochastic dynamic programming, and heuristic algorithms have been applied to solve problem matters related to water resources. Development, operation, and management are vital in a reservoir operating policy, especially when the reservoir serves a complex objective. In this study, an attempt via metaheuristic algorithms, namely the Harris Hawks Optimisation (HHO) Algorithm and the Opposite Based Learning of HHO (OBL-HHO) are made to minimise the water deficit as well as mitigate floods at downstream of the Klang Gate Dam (KGD). Due to trade-offs between water supply and flood management, the HHO and OBL-HHO models have configurable thresholds to optimise the KGD reservoir operation. To determine the efficacy of the HHO and OBL-HHO in reservoir optimisation, reliability, vulnerability, and resilience are risk measures evaluated. If inflow categories are omitted, the OBL-HHO meets 71.49% of demand compared to 54.83% for the standalone HHO. The HHO proved superior to OBL-HHO in satisfying demand during medium inflows, achieving 38.60% compared to 20.61%, even though the HHO may have experienced water loss at the end of the storage level. The HHO is still a promising method, as proven by its reliability and resilience indices compared to other published heuristic algorithms: at 62.50% and 1.56, respectively. The Artificial Bee Colony (ABC) outcomes satisfied demand at 61.36%, 59.47% with the Particle Swarm Optimisation (PSO), 55.68% with the real-coded Genetic Algorithm (GA), and 23.5 percent with the binary GA. For resilience, the ABC scored 0.16, PSO scored 0.15, and real coded GA scored 0.14 whilst the binary-GA has the worst failure recovery algorithm with 0.09. Furthermore, the reservoir optimisation using machine learning approaches was anticipated3, beginning with linear optimisation, then dynamic optimisation, and eventually heuristic algorithms, especially the Genetic Algorithms (GA). However, the authors of this study assert that these algorithms have their constraints due to the convergence speed and curse of dimensionality. To address these shortcomings of the previous methods, meta-heuristics algorithms (MHAs) were implemented for optimal reservoir operation in terms of standard operating system6 and management to solve the optimal nonlinear, nonconvex relationships and complicated problems with difficult and complicated thresholds7. The following paragraph mentioned a few of the recent MHAs been utilised in the different reservoir operation functions.Growing water demand would strain most reservoir storage systems. Optimising reservoir activities can increase water storage to alleviate this issue. More recent review in reservoir optimisation can be found in Ref.8. The adoption of the evolutionary algorithm in resolving the reservoir optimisation operation has been demonstrated in Ref.9. Meanwhile, the hybridisation of coral reefs algorithm and machine learning to solve the multi-reservoir system operation has been presented in Ref.10. From 2010 to 2019, the investigations of the time series data for optimising hydropower activity at the Karun-4 reservoir in Iran has been conducted in the studied found in Ref.11. This study demonstrated the evolutionary algorithm of GA, swarm-based algorithms of particle swarm optimisation (PSO), and a new nature-inspired swarm-based algorithm, the month swarm algorithm (MSA), used in reservoir hydropower optimisation and the reservoir simulation outcomes showed that the MSA algorithm excelled over the GA and PSO algorithms. Further on, the comparison of the operation management of the single hydropower reservoir between the Jaya Algorithms and other MHAs based on the hedging policy has been demonstrated in Ref.12. Although the Jaya algorithm had outperformed other MHAs in terms of achieving better solutions, it loses out in terms of convergence speed when compared to the PSO. Regardless of what has been interpreted above, as stated in the No Free Lunch Theory (NFL), as in any optimisation issue, there is no algorithm that can perfectly measure up to all of it13].The implementation of reviews, as well as their disadvantages and benefits with different MHAs to optimise hydropower energy production has been highlighted in Ref.14. Hence, this paper is an extension of the whale optimisation algorithm and the Lévy flight distribution conducted at the KGD by Ref.15. The following section describes the HHO algorithm, OBL-HHO, study area, and dataset. The study flowchart is also presented. Optimal KGD release curves have also been plotted. The suggested MHAs are compared to other published heuristic algorithms in terms of algorithm performance and reservoir risk analysis. Statistical errors were also evaluated. This work optimised the KGD operations with multi-objectives by utilising the HHO algorithm and OBL-HHO approaches to minimise water deficit. Lastly, the novelty of this work has been highlighted in earlier statement of this paragraph and the results have been compared to other previous heuristic algorithms in terms of reservoir risk analysis, in order for the stakeholder to consider which criteria of the reservoir risk analysis to be selected, correlated to the algorithms without jeopardising the operations of the KGD and downstream activities.As such, more work is needed to prove the efficacy of the Harris Hawks Optimisation (HHO) and the Opposite Based Learning (OBL-HHO) algorithms and these algorithms have yet to be applied at the Klang Gate Dam (KGD), even though the HHO has been applied in reservoir irrigation management which can be found in Ref.16, and its major objective is to deliver water to the two water treatment plants (WTPs) located at Wangsa Maju and Bukit Nanas districts in the Kuala Lumpur City region.The Klang Gate Dam, often known as the KGD, is located in the Taman Melawati neighbourhood of Kuala Lumpur's Gombak district. It is also known as the Bukit Tabur Dam, and the numbers 3217002 and 3217004 belong to the rainfall stations located on site . The dam was constructed at a latitude of 3° 13′ 58″ North (3.233°) and a longitude of 101° 45′ 0″ East (101.75°). The characteristics of the KGD is outlined in Ref.[Source: Lembaga Urus Air Selangor (LUAS) or Selangor Water Management Authority]. The inflow was classified into three categories, namely high, medium, and low flows, which can be found in Ref.15. According to Ref.15, Puncak Niaga (M) Sdn Bhd is the company that is involved with the running of the dam operations. The HHO and OBL-HHO were utilised to optimise the monthly reservoir release operation at the KGD. This was accomplished with reference to the monthly observed dataset of inflow, demand, storage, and losses . The optimisation process with the datasets mentioned, was performed with the Python (Anaconda) software.Observed monthly Storage, inflow, evaporation rate, and release data were collected for the years 2001 to 2019 Minimisation of water deficit.Z described as the deviation between water demand and water release, as defined by Eq. , respectively; Equation  gives thThreshold of inequality.(i)The Reservoir Storage Capacity Threshold is as follows, MCM (million cubic meter):(ii)The following is the release threshold:Penalty functions.By penalising the objective function using equation as shown below able to handle the reservoir capacity constraint.18. In which, Y is the fitness recursive objective function.The empirically determined values of the two penalty coefficients represent the tolerance of the objective function for its ultimate value, and this has been involved the flood risk reduction as of the storage is the main factor in a reservoir. As a result, Eq.  has been19. The following are the descriptions of the exploration and exploitation phases of the HHO's20.Predators with intelligence and distinction, such as the Harris hawk, can hunt, encircle, flush out, and kill prey in groups, such as rabbits. The population of hawks is considered to be a group of hawks that employ seven distinct killing tactics to chase the target rabbit (the answer to the optimisation problem). Initially, the head of the hawks will attempt to catch the prey. If the prey's movement and ability to escape make it impossible for the head of hawks to catch the prey, the group will switch tactics. The remaining members (the hawks) will continue to hunt the prey until it is killed. Hawks are able to hunt the escaping prey by confounding and exhausting it. In the HHO, Harris hawks are classified as candidate solutions, whilst the intended prey is defined as the optimal or global solution. Consequently, the HHO undergoes exploratory and the zigzag motion of the prey during the escaping initiated the Lévy flight distribution character in Harris Hawk with short length jump and boost the searching strategies in exploitative phasesmin and Smax, was determined by using N as the state option, and the resulting value was then segmented into N − 1 intervals. As a result, the discretisation of the storage problem has been resolved and is shown in Table Figure 21. The main idea behind the OBL is to search for both an estimate and its opposite estimate at the same time, in order to obtain a better estimate of the present candidate solution. The study was demonstrated by Ref.22, who applied the OBL and other integration of the enhancement techniques to solve the real-world that involved medical datasets. The OBL is an effective technique for exploring issue space more thoroughly. By simultaneously searching for the solution and its inverse, this approach can help increase efficiency. While this method increases the computing strain on the algorithm, it however significantly speeds up convergence. The following equation presented the OBL strategy in D-dimensional space. The simplified flow of the OBL for this study's investigation at KGD is represented in Fig. u,l]; The Opposite-based learning (OBL) concept was originated by Ref.d 10) belowThe root mean squared error (RMSE) is the square root of the mean of all errors squared. The RMSE is widely used, and it is regarded as an excellent general-purpose error metric for statistical evaluation.i;t; t.Because of its very intuitive interpretation in terms of relative error, the mean absolute percentage deviation (MAPD) is usually expressed the accuracy of the ratio by defined as Eq. . It is a23. The definitions of these three indices in measuring performance levels are mathematically expressed as Eqs.  were used as Eqs. –14). Th. Th23. Tinto Eq. . PeriodiThe most important index for checking the model’s performance to meet the goals during a reservoir optimisation process is resilience. The ability of a model to recover from a series of subsequent failures is another definition of resilience. The higher the resilience index, the faster the system recovers during the difficult period.Vulnerability is an expression of the degree to which a model’s failure criteria are met. The lower the vulnerability index, the more robust the system is. The expressed equation is as follows:(SI) conveys knowledge about both the periodical reliability and severity of the deficit. Equation ; Dt is the targeted demand for any month t; Rt is the water release for any month t.The shortage index Equation  calculatFigure Figure The ideal release choice for each storage type was found for each month of the year using the HHO and OBL-HHO algorithms developed, as seen in Fig. Figure Figure Figure Table Figure Table 25. The Taguchi technique uses orthogonal arrays to calculate the minimum number of experiments required to get all of the factors impacting the output parameter. The orthogonal array performed in this subsection of L16 (24) is aimed at exploring the influence of 4 factors with 2 levels of values consisting of an upper and lower boundary in 16 experiment runs. The primary effect plot for the simulated releases is depicted in Fig. Using the Taguchi model, the optimal parameters for the algorithms can be determined efficientlyA univariate ANOVA was used to investigate the impact of factors on the dependent variables using the measured data from the Taguchi method's analysis of mean variance. The findings of the univariate ANOVA for the impact of the various factors on the independent variables and the interactions between the factors by one to one are depicted in Table N is the total number of rows; k is the total number of columns; Numerous non-parametric tests, such as the Sign test and the Wilcoxon-Signed-ranks test, can be employed to handle pairwise comparisons. However, in this study, the Friedman test is used to compare the outcomes of the evaluation of the suggested algorithms' exploration and exploitation capabilities, respectively. The Friedman test was chosen since it was used to compare more than two algorithms. If there is statistically significant difference, hence, post-hoc methods such as the Bonferroni-Dunn procedure will be applied. This can be checked by adopting the following equations for Friedman test and Chi-square, respectively as below:df was calculated as the degree of freedom by subtracting the k − 1, which was equal to 2. Chi-square values of X2 = 5.3 and − 6.3 were obtained. The critical Chi-Square value obtained from the Chi-Square distribution table is 5.991 . Both X2 values obtained fell below the critical Chi-Square distribution table, indicating that there is no statistically significant difference. As a result, no post-hoc analysis was needed.According to Friedman's average rankings, the most efficient algorithm obtained is the HHO. The statistically significant difference was determined using the Chi-Square distribution table, 26. Then, in the study, the authors had utilised meta-heuristic algorithms, namely the Artificial Bee Colony (ABC), Particle Swarm Optimisation (PSO), and Genetic Algorithm (GA) carried out at the KGD. This section of our analysis compares their findings to our suggested HHO and OBL-HHO algorithms, using the similar duration and method of inflow state calculation as found in Ref.26. In this section, the performance indices are measured using million gallons (MG) as the unit of measurement. As explained in “27 to convert the amount of rain during that time period into inflow are explained in the following equation. In his study, the 50% of the total rainfall amount goes into the KGD’s catchment area.I is the inflow of the month t.The total of 22-year rainfall data beginning from year 1987 to 2008 were obtained from an investigation conducted by Ref.(MR) for the overall MHAs is presented in the final section of this Scenario II to provide a better illustration of the performance evaluation index of the algorithms28.n is the number of evaluation index in Scenario II, n = 4.The benchmarking to the category of the inflow is the same as earlier Scenario I. The findings of the performance in reliability and risk analysis for HHO, ABC, PSO, and GA are represented from Figs. 26, the ABC outcomes satisfied demand in 61.36%, 59.47% in PSO, 55.68% in real-coded GA and 23.5% in binary GA. For the excess release (surplus period), which resulted in wastage, the HHO achieved 12.88%, OBL-HHO exceeded with 15.91%, while ABC and PSO both attained similar reliability of 12.1%. However, the GA in binary generated the most wastage at 28.4%, while the GA in real-coded form generated a 14.4%. The sequence for the optimal release policy's shortage period started with the worst placement, where the binary GA, real-coded GA, PSO, OBL-HHO, ABC, and finally the HHO with the percentage of 48.10%, 29.92%, 28.41%, 27.65%, 26.54% and 23.86%, respectively.The overall performance of several meta-heuristic algorithms at the KGD is depicted in Fig. The vulnerability index is depicted in Fig. Figure Figure Overall, in scenario II, the HHO algorithm had performed well in terms of reliability, shortage index, and resilience. However, HHO failed to meet the vulnerability index criteria. Despite this, the HHO was able to present the resilience index by demonstrating that it is the most capable algorithm among other MHAs for recovering from failure. The binary-GA demonstrated the best performance in vulnerability index criteria; however, the other evaluation indexes demonstrated poor performance, particularly in reliability and resilience. According to Table This study was pursued with the aim of determining the optimal release operations at the KGD using the HHO and OBL-HHO algorithms, to minimise the water deficit. The overall findings for Scenario I demonstrated the HHO algorithm met 38.60% of demand for medium inflows. High inflows could meet 13.16% of demand, and low inflows, 3.07% of demand. With the OBL-HHO, the demand for medium inflow was met at 20.61%, then for high inflow at 9.21%, and for low inflow at 2.63%. The HHO scored 0.34 for medium inflow, 0.63 for high, and 1.23 for low. The OBL-HHO scored 0.20, 0.49, and 0.96 for medium, high, and low inflows, respectively. The final session of the evaluation used a response graph to determine how significantly a variable affects the reservoir release operation at the KGD. For Scenario II, monthly release curves from 1987 to 2008 were used  and other heuristic algorithms, such as the ABC, PSO, and GA (reported in an earlier study by Hossain (2013)). The HHO performed 62.26%, ABC 61.36%, PSO 59.47%, the OBL-HHO 56.44%, real-coded GA 55.68%, and binary GA 23.5% in reliability performance. In terms of vulnerability indices (in MG), the binary GA scored 136.91, the OBL-HHO scored 137.83, and the HHO scored 160.10. The HHO approach used in the optimal release reservoir operation recovered from a failure better than the OBL-HHO, gaining 1.56. Thus, HHO performed higher in reliability, resilience, and scarcity index.Based on the results of both case studies, the HHO algorithm is not the overall best algorithm among the other MHAs tested in this study, if all the parameters are considered together at once. Nevertheless, the HHO remains a convincing algorithm for developing monthly release curves, as evidenced by the convincing results obtained in reliability and resilience index, whereas the OBL-HHO is capable of minimising the water deficit at the KGD for the respective monthly release curves during the end storage level phase. Hence, it is up to the reservoir stakeholder to decide the algorithm best correlate to the respective climate condition. In a nutshell, future research will likely examine the impact of climate change on KGD reservoir release operations under various climate scenarios.


Background: While the prevalence of heart failure with preserved ejection fraction (HFpEF) has increased over the last two decades, there still remains a lack of effective treatment. A key therapeutic challenge is posed by the absence of animal models that accurately replicate the complexities of HFpEF. The present review summarizes the effects of a wide spectrum of therapeutic agents on HF. Methods: Two online databases were searched for studies; in total, 194 experimental protocols were analyzed following the PRISMA protocol. Results: A diverse range of models has been proposed for studying therapeutic interventions for HFpEF, with most being based on pressure overload and systemic hypertension. They have been used to evaluate more than 150 different substances including ARNIs, ARBs, HMGR inhibitors, SGLT-2 inhibitors and incretins. Existing preclinical studies have primarily focused on LV diastolic performance, and this has been significantly improved by a wide spectrum of candidate therapeutic agents. Few experiments have investigated the normalization of pulmonary congestion, exercise capacity, animal mortality, or certain molecular hallmarks of heart disease. Conclusions: The development of comprehensive preclinical HFpEF models, with multi-organ system phenotyping and physiologic stress-based functional testing, is needed for more successful translation of preclinical research to clinical trials. Heart failure with preserved ejection fraction (HFpEF) has recently been recognized as a complex clinical syndrome characterized by alterations in various organ systems. Currently, HFpEF affects nearly five percent of the general population aged ≥60 years and accounts for approximately half of the total HF hospitalizations . MoreoveHFpEF presents a major challenge for translational and therapeutic preclinical and clinical research. It is characterized by a “preserved” i.e., 50%) left ventricular (LV) ejection fraction (LVEF), abnormal LV filling, and elevated filling pressures. The pathophysiology of the HFpEF disease is multifactorial. Despite “normal” ejection fraction, HFpEF is characterized by diastolic function, characterized by impaired LV relaxation and/or increase in LV stiffness, cardiac reserves, systemic and pulmonary vascular function, renal function, oxygen carrying capacity, and peripheral oxygen extraction  Such het0% left vTreatments for HFpEF have been qualitatively summarized and discussed in a previous review  emphasizThe present review summarizes the effects of a wide spectrum of therapeutic agents on heart failure. LVEF remains a key consideration when identifying appropriate animal models of HFpEF, and special attention has been paid to its potential influence on the efficacy of candidate therapeutic interventions. More specifically, the following questions were addressed: (a) What treatment goals for HF were achieved in relation to individual potential candidates to treat the disease? (b) What animal models were chosen to evaluate these effects? (c) Could these experimental approaches mimic human HFpEF? (d) Could they provide the reproducible results given by individual medicine agents? (e) What were the factors that could influence the therapeutic efficacy observed in a particular animal model? These may well play an important role in identifying critical obstacles to therapeutic innovation in HFpEF based on preclinical investigation.In total, 10,526 articles were searched, and 664 papers were found to be potentially relevant to the review question. Of these, all protocols reporting the ejection fraction values for at least the Vehicle and Sham groups were included in the final analysis (N = 194 studies and 239 interventions), as presented in The evaluation of risk of bias is summarized in p > 0.05) test result and low possibility of missing studies (trim and fill procedure) seem to indicate the absence of publication bias across the majority of studies.The results of the publication bias analysis are presented in The review included a total of 194 studies and 239 interventions. The term intervention indicated a separate comparison between the Vehicle group (subjects with HF receiving placebo) and healthy animals, or between the Treatment group (subjects with HF receiving therapeutic agent) and Vehicle . The majP/dtmin, tau, and E/A), passive stiffness , left ventricular end-diastolic dimension (LVEDd), pulmonary edema/lung congestion, as well as LV structure , and fibrosis.Of the 239 interventions, 218 (81.3%) examining various animal models of heart failure gave baseline values of ejection fraction equal to or above 50%. In general, the baseline LVEF in this subgroup of animals was 67.69 . The second subgroup included animals with a mean LVEF below 50%, i.e., 41.58 . P/dtmin, tau, and E/A; passive stiffness: LVEDP, end-diastolic stiffness; LVEDd; LV hypertrophy) were calculated to differentiate between animals with heart failure receiving placebo (Vehicle) and healthy animals.In most cases, the models proposed for the assessments of therapeutic agents for HF met the criteria to develop the disease based on the most common comorbidities associated with human HFpEF, viz. hyperglycemia and obesity.P/dtmin, tau, and E/A), passive stiffness , left ventricular end-diastolic dimension (LVEDd), pulmonary edema/lung congestion, as well as LV structure , and fibrosis, as well as systolic function . Other studies have also found the tested agents to have beneficial effects on glucose oxidation, inflammation, oxidative stress, apoptosis and mitochondrial function, as well as renal function, systolic pressure and exercise capacity, all of which are hallmarks of the deleterious functional and structural consequences of heart failure.p < 0.05).The median duration of particular drug administration was six weeks . A meta-regression analysis of the relationship between treatment duration and resultant efficacy of several therapeutic agents is presented in The analyses mentioned in this P/dtmin, tau, and E/A), passive stiffness , left ventricular end-diastolic dimension (LVEDd), pulmonary edema/lung congestion, as well as LV structure , and fibrosis. In most cases, animals with lower baseline LVEF values tended to respond better to the tested medication.p < 0.05). The analysis comprised the same list of HF-related parameters given above , as well as metabolic syndrome models (ZSF1 Obese). These models were used for the evaluation of more than 150 different substances, predominantly SGLT-2 inhibitors, ARB, HMGR inhibitors, calcium sensitizers, DPP-4 inhibitors, and GLP-1 receptor agonists.The present paper compares the efficacy of medical interventions tested in several rodent models of heart failure (HF). A wide spectrum of protocols were evaluated. These included pressure overload  and systemic hypertensive models , type 1 or 2 diabetes mellitus  in their respective cardiovascular outcome trials, though some have shown potential improvements regarding secondary outcomes ,204. TheRecent findings have allowed the design of algorithms for multidimensional modeling of HFpEF in preclinical studies. According to this approach, the reviewed protocols were intended to assess their potential to develop an ejection fraction equal to or higher than 50%, diastolic dysfunction; exercise intolerance; pulmonary edema/congestion, and concentric cardiac hypertrophy . While aIn addition, alterations in lung mass were reported only in a quarter of all experimental protocols. Pulmonary edema/lung congestion were noted in ZSF1 Obese, Dahl salt-sensitive, SHR subjects, or those infused with angiotensin, or DOCA salt; however, this observation has not been confirmed in other protocols due to lack of data. Even fewer experiments included an assessment of the deterioration of exercise capacity. This phenomenon made it difficult to assess the degree to which a particular model used for further evaluations of effective treatments for HF could reflect human HFpEF.In more than 20% of all reviewed protocols, Dahl-salt sensitive subjects exposed to a high salt diet (8% NaCl) were used to evaluate a large number of pharmacological agents . In addition, previous findings ,7 indicaTreatment goals for patients with heart failure (HF) focus on reducing HF symptoms, improving functional capacity, enhancing quality of life, delaying the progression of the disease and reducing cardiovascular mortality. The preclinical research has mainly focused on the normalization of hemodynamic and echocardiographic parameters. Overall, the candidate therapeutic agents included in the current review improved diastolic function. The most comprehensive effect was revealed for NEP inhibitor/AT1 receptor antagonists . A lesser effect was noted for incretin-based medications , biguanides, statins and various plant-derived components. The majority of therapeutic agents normalized left ventricle hypotrophy and fibrotic lesions. While AT1/NEP inhibitors, DPP-4 inhibitors, HMGR inhibitors, MR antagonists, or PPAR-gamma agonists were all found to significantly normalize pulmonary edema/lung congestion, this parameter was assessed in only 49/194 (25%) papers.Among the reviewed models, only SGLT-2 and PDE inhibitors demonstrated a beneficial impact on animal exercise capacity in isolated studies. The latter feature seems to be important when considering translational research in this area. While many clinical studies of cardiovascular diseases, including HFpEF, have incorporated exercise testing to quantify impairments in physiologic reserves, the protocols for preclinical studies included only assessments of resting cardiac function. Some authors emphasize the need for combining cardiac imaging or hemodynamic monitoring with exercise testing. This approach would provide an insight into the physiological reserves of the animals, and such human HFpEF symptoms as dyspnea on exertion . SimilarAn analysis of mortality, a key outcome in clinical trials of effective treatments for HFpEF, was performed only in 29 papers (14.9%). Even fewer concerned the incidences of ventricular arrhythmias that could contribute to sudden death as the most common mode of death in subjects with HFpEF. Candidate drugs  improved the survival of Dahl-sensitive rats, or subjects with HF caused by pressure overload by AAB (TAC). These approaches revealed significantly poorer survival compared with diabetic (obese) subjects, but did not reflect human HFpEF due to the transition from compensated hypertrophy to congestive heart failure . On the P/dtmin)—in relation to BW gain and/or higher glucose levels. In addition, elevated systolic blood pressure was found to have a significant impact on LV hypertrophy. These phenomena concerned ZSF1 Obese animals that manifested a significant increase in body weight , and hyperglycemia . The potential of the model to develop a disease as a result of these conditions appears to confirm its appropriacy for evaluating drug efficacy. However, only a relatively small number of experimental protocols have been performed on this model in order to identify candidate intervention for HFpEF (<10% of the reviewed papers), and this can be seen as a limitation of the study.The presence of pathologic lesions in human HFrEF is largely driven by a primary defect in systolic function. Such dysfunction can be induced in animals with clinically relevant interventions, e.g., by coronary artery ligation . In contAs discussed above, when developing HFpEF in animals, the individual experimental protocol should result in the preservation of an ejection fraction of at least 50%. As such, papers that did not report data about LV ejection fraction (N = 111) were excluded from the analysis. More than 80% of experimental approaches displayed baseline ejection fraction values of at least 50%. These animal models were consequently included in the final analysis of the potential therapeutic efficacy of medicinal agents on HFpEF.Interestingly, in the Vehicle group, it was possible to differentiate animals according to their potential to develop the disease and the degree to which they responded to tested agents by assuming a threshold baseline ejection fraction of 50%: for the majority of parameters, EF < 50% was associated with the development of more deleterious diseases. As a consequence, at lower baseline LVEF% values, the candidate therapeutic agents demonstrated greater disease normalization with regard to various aspects of HFpEF, including active relaxation, left ventricular end-diastolic dimension, pulmonary edema/lung congestion, or left ventricle hypertrophy. In a particular model, in untreated subjects with HF, preserving LVEF seems to favor the acquisition of reproducible and repeatable results for individual medicines.Animal models provide a valuable insight into the underlying molecular mechanisms of the disease. This phenomenon might concern HFpEF, a multisystem disorder that also involves the kidneys, skeletal muscle, adipose tissue, and immune and inflammatory signaling . ActivatAs demonstrated above, individual pharmacological agents had different effects on LV ejection fraction, the most commonly reported parameter, according to particular animal models. However, due to the limited number of studies addressing particular drugs , the variety of HF models used in the experiments, as well as the wide spectrum of the reported parameters, it was not possible to make similar comparisons to those made for LVEF%. Such comparisons might include the influence of an individual therapeutic agent (class) on the normalization of a particular parameter associated with diastolic dysfunction, according to the animal model. This phenomenon is one of the main limitations of the present study.Another limitation is that, due to the limited number of experiments, our analyses did not include any evaluation of the potential advantages of two-hit approaches with regard to therapeutic interventions, e.g., high-fat diet plus ApoE−/−; Ang II infusion; L-NAME; or STZ, or combinations such as ZDF plus aortic constriction or Zucker fatty rat plus 6%NaCl. This requires further study. In addition, the efficacy of combined treatments for HF were not studied in these models for the same reason.The PubMed and Web of Science databases were searched for papers published from January 1992 to May 2022. The search encompassed experimental protocols investigating the influence of medicinal agents on the improvement of heart failure, with special attention paid to heart failure with preserved ejection fraction, in animal models of the disease. Only protocols specifying LVEF were included, as this has been used to classify subjects into HF with preserved ejection fraction in a range of preclinical and clinical settings . A comprpopulation); drug, route of administration, dosage, treatment duration (therapeutic intervention); placebo control, other medicine agent (comparator); and outcomes. The extracted outcome data included body weight (BW) gain (loss), left ventricle (LV) weight, LV weight/BW, LV weight/tibia length (TL), lung weight, lung weight/BW, lung weight/tibia length (TL), lung wet/dry ratio. The laboratory parameters included blood glucose (GLU), insulin (INS), total cholesterol (TC) and triglyceride (TG) level. The following hemodynamic data were recorded: systolic blood pressure (SBP), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and time needed for relaxation of 50% maximal left ventricular pressure to baseline (tau). Echocardiographic parameters included left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), ratio of E-wave to A-wave (E/A), maximal rate of pressure rise (dP/dtmax) and pressure fall (dP/dtmin). The degree of LV fibrosis  were extracted as histopathological data, as described above [The recorded data included, according to the PICO acronym, animal age at the start of experiment, sex, species, background strain, characteristics of HF model , another cohort of healthy animals (Sham) and another cohort receiving particular therapeutic agents (Treatment). The literature was searched by independent reviewers , who extracted the data and assessed the risk of bias. Any disagreements were solved by discussion until consensus was reached.The risk of bias was assessed using the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias tool for animal studies . An EggeHF+placebo), and healthy animals  were expressedThe effect size for the Vehicle group was calculated with a 95% confidence interval (CI). Changes in the mean response (X) given by the Vehicle group :HF+drug) and the Vehicle group  were expressedThe effect size for the Treatment group was calculated with a 95% confidence interval (CI). Any differences in the mean response (X) given by animals with HF receiving therapeutic agents :Every comparison between Vehicle vs. Sham, or Treatment vs. Vehicle was regarded as a separate ‘intervention’.worsening of the disease), while a decrease in D indicates a reduced value of a particular parameter in the Treatment group as compared to the Vehicle group .In most cases, changes in echocardiographic, hemodynamic parameters, as well as in animal body weight or laboratory parameters were analyzed using Formula (1) for the Vehicle group, or Formula (3) for the Treatment group. An increase in D (difference in means), indicates a rise in the value of a particular, HF-related, parameter in the Vehicle group as compared to healthy subjects  and healthy rodents were calculated using Formula (2), and differences between Vehicle and Treatment group were calculated using Formula (4). A response ratio (R) of 1 denotes no dissimilarities between Vehicle and healthy subjects, or between the Treatment group and Vehicle group. Values R > 1 (R < 1) designate an increase (or a reduction) in a particular parameter in a respective HF model . As the p-value < 0.05.Meta-regression and subgroup analyses were performed where possible to evaluate the influence of particular variables  on the outcome. The statistical analyses were performed using a random-effects model (STATISTICA 13.1). Heterogeneity between two (or more) animal subgroups was indicated by a statistically significant Cochran’s Q score. A significant result was indicted by a There remains a need for comprehensive preclinical HFpEF models employing physiological stress-based functional testing approaches that can mimic human diseases characterized by a multi-organ phenotype; this need is particularly pressing considering the growing incidence of HFpEF in aging populations and the lack of targeted therapies. Existing preclinical studies in HFpEF have identified a wide spectrum of candidate agents that have been found to be effective against LV diastolic performance. Their activity has been evaluated in a variety of experimental protocols, most of which are based on pressure overload or systemic hypertension, with a smaller number examining type 1 or 2 diabetes mellitus, or obesity; however, some of these models could fail to reflect human disease.(a)Experimental approaches are characterized with varying degrees of effectiveness regarding their ability to develop the disease by the most common comorbidities associated with human HFpEF . The ZSF1 model is able to develop disease by a similar set of conditions to humans; as such, it appears suitable for identifying candidate medications.(b)The minority of experiments concerned drug-related normalization of pulmonary congestion, and even fewer entailed the improvement of animal exercise capacity. Both features represent treatment goals for patients with HF, and this phenomenon can make it difficult to comprehensively evaluate effective treatments for human HFpEF.(c)The baseline ejection fraction can play a key role in determining the extent to which particular medicine agents normalized the disease according to diastolic dysfunction, LV hypertrophy, or pulmonary congestion. In a particular model, preserving LVEF seems to favor the acquisition of reproducible and repeatable results for individual medicines.(d)The mortality associated with a candidate agent can be determined based on the likelihood of an individual experimental model to worsen animal survival. This should be taken into consideration in studies comparing different therapeutic agents.(e)Animal models of HFpEF might represent a promising approach for evaluating the deleterious functional and structural consequences of the disease at the molecular level, and for identifying future specific drug therapies. However, these effects were reported in only a small number of papers.In contrast, fewer studies have assessed pulmonary congestion, exercise capacity, arrhythmia, and animal mortality, as well as certain molecular hallmarks such as inflammation, oxidative stress and endothelial dysfunction. This gap may partially explain why traditional medical agents have been found to demonstrate negative or neutral effects in clinical trials, despite having positive outcomes in experimental studies. Other potential explanations may be:


In the IGNR system, reline consisting of choline chloride and urea acted as both a solvent and a nucleophile generator, and the nucleophilic addition–elimination mechanism was verified by model compound studies. The in situ generated NH3 could precisely cleave the lignin–carbohydrate esters in lignocellulose with a near-quantitative retention of carbohydrates. The extracted IGNR–Protolignin exhibited native lignin substructure with high molecular weight and high β-O-4′ content (41.5 per 100 aromatic units). In addition, the up-scaled kilogram reaction demonstrated the feasibility of the IGNR system for potential industrial application in a green and sustainable pathway. This work represents a breakthrough toward protolignin extraction in practice with the future goal of achieving total biorefinery.Protolignin extraction can facilitate structure elucidation and valorization of lignin in biorefinery, but is rather challenging due to the complex chemical bonds present. Here, we developed the in situ generated NH Lignin, along with cellulose and hemicellulose, is a major component of the plant skeleton and contains unique aromatic building blocks. It is the second-largest amount of plant-based organic matter only after cellulose and the most abundant natural aromatic polymers –3. HowevCurrently, only several protolignins or protolignin derivatives have been prepared, and relevant research is still in progress. Regarding protolignin extraction, milled wood lignin (MWL) was first proposed by Björkman in 1954 , and is Protolignin derivatives (or native-like lignin) can also be extracted from lignocellulose using a “stabilization” strategy. For example, acidic catalysts or relative higher temperatures are applied to promote lignocellulose fractionation and lignin extraction, while stabilized molecules in the system are grafted onto the lignin, protecting it from subsequent depolymerization or the formation of active intermediates . A repreTo solve the above problems, we summarize the structure–activity relationships of the well-known lignin–carbohydrate complexes (LCCs) at the molecular level Fig. A. Lignin3-reline (IGNR) system was developed for efficient protolignin extraction  that generated by partially sacrificing urea acts as a nucleophilic reagent to attack the carbonyl carbon atom in the lignin–carbohydrate esters. By precisely cleaving the lignin–carbohydrate esters through IGNR, the liberated IGNR–Protolignin is subsequently solubilized in the solvent and can be readily isolated, simultaneously providing the carbohydrates as an insoluble residue in nearly quantitative yield. In this study, the relevant trial was first carried out by model compound studies to verify this hypothesis. Then, the chemical structure and molecular weight of IGNR–Protolignin were thoroughly investigated and compared with MWL . Finally, the reaction was up-scaled 100-fold to reveal the potential industrial application of this strategy. Further prospects for entirely closed-loop lignocellulose refinery based on the system were also discussed, including solvent recovery and reuse, and by-product immobilization as fertilizer. Overall, this work demonstrated a new route for protolignin extraction and promoted the development of future all-component biorefinery.Here, we developed a strategy for one-pot protolignin extraction from lignocellulose. A green and low-cost in situ generated NHion Fig. B. It sho− in high-temperature alkaline aqueous solutions has a hydrolytic effect on hemicellulose as well as the ability to nucleophilically cleave (non-)phenolic β-O-4′ bonds in the lignin structure  would form. NH3 acts as a nucleophilic reagent to precisely cleave the lignin–carbohydrate esters via nucleophilic addition–elimination. In this process, NH3 as a gas nucleophile demonstrates obvious advantage compared with other solid or liquid nucleophilic reagents in terms of accessibility to the cell wall of lignocellulose. Meanwhile, the gas nucleophile would have minimal effect on the carbohydrate decomposition compared with other aqueous or organic solvent-based systems. The liberated IGNR–Protolignin tends to dissolve in the hydrogen bond-rich reline system prior to lignin isolation.We thus present an IGNR system for selective cleavage of lignin–carbohydrate esters and protolignin extraction Fig. B. Typicad-glucopyranoside (1b), and benzyl benzoate (1c) as a lignin aryl ether bond, lignin–carbohydrate ether bond, and lignin–carbohydrate ester bond, respectively. For a typical IGNR reaction, the as-prepared reline solvent and model compounds were mixed and sealed in an autoclave and heated to 150 °C (starting decomposition temperature of urea), which was maintained for 6 h for continuous NH3 generation.To verify the feasibility of the IGNR system for targeting cleavage of lignin–carbohydrate esters, several control experiments were performed using model compounds, including 2-(2-methoxyphenoxy)-1-(4-methoxyphenyl) ethanol (1a), phenyl β-3), almost no conversion of 1c was observed, revealing the specific impact of NH3 on the lignin–carbohydrate ester in the IGNR system. Based on the product distributions from the model compound studies, we propose that the mechanism for selective cleavage of lignin–carbohydrate ester was mainly achieved through nucleophilic addition–elimination . At the same time, the removed benzyloxy group was alkaline enough to obtain protons, forming the major product of 2c, while the minor product 4c was likely formed from the reaction of 2c and urea.It was observed from the gas chromatography (GC) results that only 1a was detected after the reaction, demonstrating the good stability of lignin β-O-4′ linkages in the IGNR system Fig. A. When 13 gas attacked the lignin–carbohydrate ester in lignocellulose, and the liberated lignin fragments were solubilized in reline for extraction. This allowed the system to precisely cleave the lignin–carbohydrate esters in the LCCs while simultaneously establishing a protective system for the ether bond in the lignin, achieving one-pot protolignin extraction from lignocellulose.In summary, these verifications by model compounds demonstrated the mechanism for protolignin extraction via the selective cleavage of lignin–carbohydrate ester Fig. E. IGNR wBased on the studies using model compounds, one-pot protolignin extraction from poplar wood sawdust was carried out in the IGNR system at 150 °C for 6 h with a 1:10 mass ratio of poplar sawdust Fig. A to solvCompositional and functional group analyses of IGNR–Residue were carried out to verify the effect of IGNR fractionation on the carbohydrates . From th−1 and the C–O ester bond at 1,234 cm−1 almost disappeared after the reaction, proving that the lignin–carbohydrate ester was effectively cleaved by the IGNR system [Meanwhile, Fourier transform infrared (FTIR) analysis was performed on the raw material and IGNR–Residue. The spectrum of the raw material Fig. E shows tR system –50. In sα-Hα at δC/δH = 71.5/4.87, Cβ-Hβ at δC/δH = 85.9/4.12 and 83.9/4.29, Cγ-Hγ at δC/δH = 59.5 to 59.7/3.40 to 3.63) and in less abundant resinol substructures  and phenylcoumaran substructures . Also, both IGNR–Protolignin and MWL showed structural signals associated with the p-hydroxycinnamyl alcohol end groups . In the aromatic region, the signals of both syringyl  and guaiacyl units  were pronounced and almost free of polycondensation. Unlike MWL, the p-hydroxybenzoate substructures  showed a slightly reduced content (from 21.8/100Ar to 9.2/100Ar) in IGNR–Protolignin. This can be explained by the effective disruption of the ester bond in the PB unit during nucleophilic addition–elimination, resulting in a weakened PB signal. It should be noted that p-hydroxybenzamide was also identified after extraction of the fractionated mixture . These results were strong evidence that the IGNR system can perform the precise dissociation of lignin–carbohydrate esters.To investigate the interlinkages of IGNR–Protolignin, MWL was used as the typical protolignin for comparison. Both the samples were characterized by 2-dimensional heteronuclear single quantum coherence–nuclear magnetic resonance (2D HSQC-NMR) spectroscopy. The side-chain and aromatic regions of MWL and IGNR–Protolignin are shown in Fig. γ position of the syringyl units, leading to easier isolation of high S-ratio lignin in case of the dissociation of ester bonds [The number of linkages between the basic units was semiquantitatively calculated by HSQC. The results showed that the number of various linkage bonds in IGNR–Protolignin was close to that in MWL: the content of dominant β-O-4′ in MWL and IGNR–Protolignin was 42.2 and 41.5 per 100 aromatic units , respecter bonds ,53. Whener bonds , the resMw) of IGNR–Protolignin  was higher than that of MWL . The polydispersity index of IGNR–Protolignin was also larger than that of MWL (4.1 versus 3.2). The GPC curves of the 2 lignin species were fitted using the log-normal distribution function for peak differentiation imitation, and the results are shown in Fig. Mw values of the 2 main peaks of MWL were 1,048 g/mol (pink area) and 3,197 g/mol (blue area), and the ratio of oligomeric and medium lignin fractions was calculated as 22:78. The peak Mw values of the 3 main peaks fitted in IGNR–Protolignin were 507 g/mol (pink area), 3,015 g/mol (blue area), and 7,727 g/mol (yellow area). High-molecular-weight lignin fractions were predominated (~87%) in IGNR–Protolignin. Possible substructures of IGNR–Protolignin are proposed according to the in-depth GPC and HSQC NMR analysis as shown in Fig. The molecular weights of MWL and IGNR–Protolignin were tested separately by gel permeation chromatography  decreased a bit and extra minor condensation of signal S-type units was observed as shown in Fig. 3 at higher temperature. A further increased temperature of 180 °C led to a reduced content of β-O-4′ (27.8/100Ar) and more severe condensation in the extracted lignin, albeit the lignin yield increased to 45.4 wt%. Nevertheless, reaction temperatures of 165 and 180 °C also demonstrated high (>92%) carbohydrate retention, indicating that the IGNR system had little effect on the cellulose and hemicellulose components even at higher temperatures. Possibly other linkages such as ether bonds in lignocellulose were cleaved at higher temperature. Therefore, with the focus of protolignin extraction with native substructures, a temperature of 150 °C is relatively appropriate for the IGNR system.For the reaction condition considerations, we found that the in situ generated NHThe optimizations of reaction time were carried out at 150 °C for 3, 6, 9, and 12 h, respectively. The yields of IGNR–Protolignin were correspondingly 23.6, 31.3, 32.7, and 34.2 wt%. It was found from the results that yields of IGNR–Protolignin were comparable except that lower yield was obtained for 3 h. Therefore, 6 h was chosen as the optimal reaction time from the view of moderate yield and lower energy consumption. In summary, at 150 °C, 6 h was chosen as the optimal conditions for the IGNR–Protolignin extraction in this work. Nevertheless, it was suggested that a temperature range of 150 to 165 °C was also applicable for IGNR–Protolignin extractions.3 pressure was ~0.6 MPa. After reaction for 6 h, the mixture was cooled naturally. The separated lignin was dissolved by acetone antisolvent solution while sealed, and then the acetone was removed from the lignin solution by rotary evaporation. IGNR–Protolignin was finally precipitated by adding an appropriate amount of water. Excess generated NH3 could be converted to NH4Cl as fertilizer  and 10 kg of reline were mixed in a 15-l sealed industrial pulping reactor Fig. A. The rezer Fig. .Mw was 5,530 g/mol  substructures in the bamboo lignocellulose disappeared, further revealing the effectiveness of the IGNR system. However, it was found that the raw bamboo lignocellulose contained non-acid-degradable silica ash that could hardly be removed, resulting in a low lignin yield of 12.9 wt% after calculation. In addition, the yield of IGNR–Protolignin–wheat straw was 20.8 wt% and the lignin was not condensed, with only a small amount of pCA substructure left. On the whole, the preliminary results demonstrated promising applicability of the IGNR system on various biomasses. Additionally, it should be noted that the lignin–carbohydrate ester content varied among different lignocelluloses. The effectiveness of the IGNR system when using other lignocelluloses still requires further study, especially for lignin–carbohydrate ester-rich or genetically modified plant-derived lignocelluloses.For the applicability of the IGNR system on different lignocellulosic resources, additional 4 types of lignocellulose from birch, pine, bamboo, and wheat straw were used to perform protolignin extraction under the same conditions as poplar IGNR–Protolignin extraction. It was found that all IGNR–Residues displayed similar appearances. Figure 3 when the temperature increased to 150 °C. NH3 was the most important link in the system, which performed the most important task of breaking the ester bond as a nucleophilic reagent. Solid lignocellulose was dispersed in reline, and the liquid reline provides a strong hydrogen bonding system necessary to help deconstruct lignocellulose and dissolve the lignin. We specifically compared IGNR–Protolignin with MWL in terms of reaction time, lignin yield, molecular weight, β-O-4′, and monomer yield after depolymerization, and the results are shown in Table This work mainly focused on the protolignin extraction by targeting lignin–carbohydrate ester cleavage. An IGNR system was designed, and the whole system showed perfect cooperation. Although reline was simply a liquid solvent at room temperature, the urea in the solvent started to decompose to NHp-hydroxybenzoate interlinkages decreased after IGNR treatment. By a simple extraction of the recycled reline, p-hydroxybenzamide was identified using GC-MS  at 80 °C with stirring until a homogeneous clear liquid appeared.Model compound verification in the IGNR system. Three model compounds, 2-(2-methoxyphenoxy)-1-(4-methoxyphenyl) ethanol (1a), phenyl β-d-glucopyranoside (1b), and benzyl benzoate (1c) (0.1 g), were added to reline (20 g) and reacted at 150 °C for 6 h in a closed autoclave . Another reaction in an unclosed round-bottom flask was used as the control. Next, 10 g of the cooled reaction product was extracted by adding ethyl acetate  and water (15 ml), and the ethyl acetate phase was concentrated under reduced pressure and dried. The product was diluted in ethyl acetate (2 ml) through a 0.22-μm polytetrafluoroethylene filter and analyzed by GC-MS and GC-FID (Flame Ionization Detector).IGNR–Protolignin extraction. First, 10 g of poplar sawdust was added to 100 g of reline in a sealed autoclave and heated at 150 °C for 6 h with continuous stirring. After cooling to room temperature, the mixture was dissolved in a 10-fold volume of acetone and water mixture (7:3 by volume) with stirring, and the insoluble residues were filtered out and dried. Then, the mixed solution was rotary evaporated under reduced pressure to remove recyclable acetone. IGNR–Protolignin was precipitated from the remaining aqueous solution and dried at 45 °C for further characterization. MWL was extracted from lignocellulose for comparison, and detailed procedures were performed according to the literature [terature ,27.Scaled-up extraction of IGNR–Protolignin. The extraction of IGNR–Protolignin up-scaled 100-fold was performed with the same operating steps as for the initial reaction. Scaled experiments were conducted using 1 kg of poplar sawdust and 10 kg of reline in a 15-l sealed cooking reactor equipped with a pressure detector . The reactor was programmatically heated to 150 °C and reacted for 6 h. Pressurized NH3 inside the reactor was released before opening the reactor. Excess unreacted NH3 was released and stabilized in aqueous hydrochloric acid, affording ammonium chloride after concentration and crystallization.Composition analyses of the insoluble residue. The compositions of the insoluble residue were measured according to the National Renewable Energy Laboratory procedure. The monosaccharides were detected by high-performance liquid chromatography . An Aminex column HPX-87H  was used to analyze the monosaccharides at 50 °C with 5 mM sulfuric acid at 0.6 ml/min. Under these conditions, xylose, mannose, and galactose were eluted at the same retention time, and the detailed monosaccharide moieties in the residue were tested by high-performance anion exchange chromatography . The sample was treated with 6% H2SO4 at 105 °C for 2.5 h and then filtered, diluted 100-fold before the HPAEC analysis. Calibration was performed with standard solutions of d-glucose, d-xylose, d-mannose, glucuronic acid, and galacturonic acid.2D HSQC-NMR spectroscopy. First, 60 mg of lignin was dissolved in 0.6 ml of dimethyl sulfoxide (DMSO)-d6. All spectra were acquired on a Bruker AVIII HD 500 spectrometer . The spectral widths were 5,000 and 20,000 Hz for the 1H and 13C dimensions. A total of 1,024 collected points were used for the 1H dimension with a recycle delay of 1.5 s, while 64 and 256 transients were used for the 13C dimension. The DMSO-d6 peak was applied as internal chemical shift reference point (δC/δH = 39.52/2.50).GPC analysis. Dried acetylated lignin samples were dissolved in tetrahydrofuran (2 mg/ml), filtered through 0.22-μm polytetrafluoroethylene filters, and analyzed by HPLC. The columns used were PLgel 5 μm Guard (50 × 7.5 mm), PLgel 5 μm MIXED-C (300 × 7.5 mm), and PLgel 3 μm MIXED-E (300 × 7.5 mm) in tandem, and the injection volume was 20 μl. The Mw value of lignin was calculated using the polystyrene standard sample with average Mw of 640, 1,230, 1,880, 4,720, 6,580, 9,580, 22,130, and 27,500 (g/mol) as standard curves. Mw was calculated using Agilent GPC software, the GPC curve was split by Peakfit software, and the log-normal amp was used for fitting to obtain the experimental results.


The interest in silver radioisotopes of medical appeal  has been recently awakened by the versatile nature of their nuclear decays, which combine emissions potentially suitable for non-invasive imaging with emissions suited for cancer treatment. However, to trigger their in vivo application, the production of silver radioisotopes in adequate amounts, and with high radionuclidic purity and molar activity, is a key prerequisite. This review examines the different production routes of silver-111, silver-103 and silver-104m,g providing a comprehensive critical overview of the separation and purification strategies developed so far. Aspects of quality  are also emphasized and compared with the aim of pushing towards the future implementation of this theranostic triplet in preclinical and clinical contexts. When properly harnessed, the radiation emitted during the decay of unstable nuclei can be exploited for the diagnosis and treatment of cancer. In recent years, cutting-edge studies in this field delved into the exploration of new metallic radionuclides able to provide a broad plethora of diagnostic and therapeutic emissions, but also the new challenges in their production and purification.111Ag) is a medium energy β− emitter , with a 7.47 d half-life (t1/2), that could be harnessed for the treatment of medium to large tumors due to its particulate emission ranging from one to ten millimeters + and [Pd(NH3)4]2+ complexes. After a washing step with water (20 mL), necessary to remove the excess of NH3, 111Ag-containing residue was eluted with a 0.5 M NaCl solution  as [AgCl3]2−. Palladium was recovered by eluting the resin with 14 M HNO3 (80 mL). The concentration of Pd2+ in the 111Ag+ eluate was <1 μg/mL.Cation Exchange: Mansur et al. reported a cation-exchange-based chromatographic separation of d matrix . Accordid matrix . The pro process . Silver 3 and evaporated to near dryness. The concentrate solution was diluted with water to obtain a 0.1–0.5 M HNO3 concentration range and passed through a Dowex resin Al  packed in a column (0.75 cm diameter × 12 cm) − and [Pd(NO3)4]2−, which are strongly retained by the AG1-X8 resin, while Ag+ remains in a cationic form that is weakly absorbed. 111Ag+ was hence eluted in 3 M HNO3 (15 mL) and several fractions were combined and dried down under air and gentle heating. The average 111Ag+ recovery was 92.9 ± 23.7% with a Pd2+ concentration <25 ppb.An alternative method, based on anion exchange resin, was reported by Aweda et al. ,10. In ts of HCl ,10. Addi111Ag was loaded into the column. Under these conditions, Pd2+ was strongly retained by the resin while 111Ag+ was easily eluted out. Recently, Ohya et al. studied the separation of the silver radioisotopes obtained from proton- or deuteron-irradiated natPd-targets by using the same anion exchange resin (Dowex 1X8) . After 25 min, the organic phase was removed and filtered (via an inorganic Anotop 0.2 μm filter). The addition of 0.1 M acetate buffer  allowed the re-extraction of 111Ag+ at a >70% yield. The residual amount of Pd2+ was determined by inductively coupled plasma mass spectrometry (ICP-MS) and resulted in a depletion by a factor of up to 27,000 (~3 μg).Alberto et al. reported the possibility of using liquid/liquid extraction to separate matrices . Accordiic phase . On the 103Ag, 104Ag, 105Ag, 106Ag, 110Ag and 112Ag  by the α-particle irradiation of a natural palladium target in a 40 MeV cyclotron (2 μA), and their subsequent extraction and separation by using di-(2-ethylhexyl)phosphoric acid (HDEHP) as a liquid cation exchanger [3 and the solution was made ammoniacal by the addition of concentrated NH3 followed by the addition of H2O2. The solution was then shaken with an equal volume of 0.1% HDEHP/octanol solution. Cadmium radioisotopes were extracted by the organic phase due to the formation of cationic cadmium species like [Cd(OH)(H2O)x]+ and [Cd2(OH)(H2O)x]3+, leaving rhodium and silver radioisotopes, along with the palladium of the matrix, in the aqueous phase. The aqueous phase was treated with an equal volume of 10% HDEHP/0.1 M citric acid solution and the silver radioisotopes, along with the bulk palladium, were completely extracted as [Pd(H2O)4]+ and [Ag(H2O)2]+, respectively. Finally, the silver radioisotopes were stripped into 6 M HNO3 2− anionic complex, leaving the Cd, Rh and Ag radioisotopes in the aqueous phase. After palladium separation, the aqueous solution was converted to a 5 M HCl solution and Cd (100%) and Ag (90%) radioisotopes were extracted as anionic chloro-complexes by the addition of TOA. Contamination with Rh was < 5%. The extraction with 8 M HCl stripped back the Ag radioisotopes into the aqueous phase and they could be separated by Cd ones.Lahiri et al. also developed another process using trioctylamine (TOA) as a liquid anion exchanger to separate  2.3 μA) . After tA schematic illustration of the liquid/liquid extraction processes herein described is shown in 3 spiked with one drop of HCl  [111Ag recovery of 93% and a palladium removal of >99.99%.Very recently, Blackadar et al. employed precipitation processes to isolate  targets . The pal105Ag, 106Ag and 111Ag) produced by bombarding a palladium target with 19 MeV deuterons  through a precipitation technique using mercury(I) chloride as co-precipitant [3 (0.5 mL) was added and the mixture was vigorously stirred. The precipitate of Hg2Cl2, containing over 95% of the radioactive silver, was centrifuged, washed with 0.5 M HCl and dissolved in the minimum needed volume of 16 M HNO3 [2SO4 (200 mg) was then added, and the solution was evaporated to dryness  to drive off the mercury carrier. The residue was quantitatively solubilized in distilled water (10 mL) to give an isotonic saline solution of radioactive silver.Co-precipitation with mercury(I) chloride: Haymond et al. reported the separation of radioactive silver radioisotopes  was treated with aqua regia (5 mL) and the obtained solution was evaporated to dryness. HCl was added and subsequently removed by evaporation [111Ag+ trapped in the lattice, was separated as solid crystals. Crystals were filtered and washed with saturated NaCl (3 × 10 mL) to remove Pd2+ traces. In the right conditions, it was shown that traces of co-precipitated Pd2+ do not exceed 0.01% of the separated 111Ag+ activity. Finally, the authors stated that the saline solution containing 111Ag+ can be directly used for medical preparations or, alternatively, it can be readily separated by electrodeposition on a Pt electrode and subsequently dissolved in HNO3 [Micheev et al. demonstrated that position . In factporation . The res in HNO3 .111Ag by electrolysis of the palladium target solutions [+ and Pd2+ . The solution was kept at an alkaline pH and then subjected to electrolysis to attain the 111Ag+ electrodeposition upon a Pt cathode [3 and HCl (50 mL/g Pd). The nitrate ion was then removed by fuming and the excess acid was completely neutralized by adding a solution of NaOH. The complexing agent was added to this solution and the mixture underwent several cycles of electrolysis at ambient temperature. Then, a large part of the solution was removed by suction and the cell was carefully flushed with 0.1 M sodium perchlorate and water. Finally, the cathode was removed and dried for the subsequent recovery of the electroplated 111Ag. Under the proper electrolysis conditions and using 0.1 M NaCN + 1.0 M NaOH as complexing agents, a 111Ag recovery around 77% was reported.Griess and Rogers obtained carrier-free olutions . Their m cathode . Practic3. Preliminary anodic or cathodic polarized platinum wire was used as an adsorbent and was immersed into the solution and kept under constant stirring. After 30 min, 95–97% of 111Ag+ were found to be adsorbed by the platinum wire. The desorption proved to be completed after having rinsed the adsorbent with a small volume of 9 M HNO3 [Selective adsorption on platinum surface: Miller et al. reported the possibility of separating traces of radioactive silver from palladium solutions through the selective adsorption of the silver ions on a platinum surface . In the 9 M HNO3 .111Ag from Th target: Mastren et al. reported the recovery of 111Ag from a proton-irradiated thorium matrix using a solvent-impregnated CL resin composed of alkyl phosphine sulfides and alkyl phosphine oxides [3 (5 mL). Subsequently, 50 μL of the obtained solution was diluted with 10 M HCl (5 mL) and passed through a column containing the preconditioned (10 M HCl) CL resin (1 mL). 225Ac produced from irradiation and the bulk Th target were not retained by the resin, whilst 111Ag was blocked and then eluted by using 10 M HNO3 (5 mL).Separation of 111Ag+, i.e., 117mSn (6%), 95Nb (6%), 99Mo (3%), 95Zr (2%), 126Sb (2%), 131I (1%) and 103Ru (1%). Thus, the 111Ag-containing fraction underwent a subsequent fine-purification step. After evaporation and reconstitution with 10 M HCl (5 mL), the solution was passed through a DGA  extraction chromatography column (1 mL). Under these conditions, 117mSn, 95Nb, 99Mo, 95Zr and 126Sb were highly adsorbed and could be separated, while the solution  was further passed through a CL resin (250 μL) and subsequently washed with 10 M HCl (2 mL) for extracting 103Ru. Finally, 111Ag was obtained by flushing 10 M HNO3 (5 mL) through the resin. 111Ag recovery by this process was reported as being equal to 93% with a radiochemical purity of >99.9%.The process gave a 94% yield but a 79% radionuclide purity, since many radioactive contaminants co-eluted with 111Ag was evaporated to near dryness and diluted with 1 M citric acid/citrate buffer . The solution was then passed through a cation exchange column containing AG50W-X8 (15 mL) and two washing steps with 1 M citrate pH 2 (5-column volume) were followed. 225Ac was retained by the column while 111Ag was recovered in the washing solution.In the second process , the dis111Ag solution but a 103Ru contamination (<1%) was still present. The latter was hence removed by passing the solution, previously evaporated to dryness and reconstituted with 10 M HCl (5 M), through a CL resin column (250 μL). The column was then washed with 10 M HCl (2 mL) and 111Ag was eluted with 10 M HNO3 (5 mL) [The process removed the greatest part of the radionuclidic impurities from the 111Ag concomitantly with 225Ac, the time-consuming separation of the two radionuclides, as well as the presence of not negligible amount of 110mAg (t1/2 = 249.8 d), represent the disadvantages of this method [Despite the potential of producing 111Ag from 111Cd: Stable 111Cd is the main isobaric contaminant remaining when 111Ag is obtained via the isotope mass separation on-line technique (ISOL—vide infra). To efficiently separate Ag+ from Cd2+ and selectively harvest 111Ag, Tosato et al. employed an extraction chromatographic resin (CL resin) [2+ was quantitatively removed in the washing step (0.1 M HNO3) whereas Ag+ was firstly retained and subsequently eluted with 10 M HNO3. The reported recovery yield was 90 ± 5% [3 was used to quantitatively elute Ag+ instead of 10 M HNO3. Finally, 1 M H2SO4 was employed in the washing step of the third method to selectively remove Cd2+, while the elution of Ag+ was conducted using 0.1 M thiourea (yield 92%). Although these methods hold promise in depleting Cd contamination from ISOL-produced 111Ag, they have yet to be tested using irradiated samples.Separation of L resin)  and deve 90 ± 5% . In the The mass separation technique evolved from chemical techniques used to separate radioactive isotopes from irradiated targets. According to this method, the radionuclides of interest are produced by irradiating a target with a particle beam . Then, the produced radioactive species are ionized and released from the target. Subsequently, the so-generated ion beam is extracted and directed to a magnet where all the concurrently produced non-isobaric species are separated. After the mass-separation step, the isobaric radioactive beam containing the radionuclide of interest is dumped on an implantation substrate for the collection ,39.111Ag via the ISOL technique in the framework of the ISOLPHARM  project [111Ag could be produced with elevated production rates and extremely high purity  by bombarding a uranium carbide target with a 40 MeV proton beam (200 μA) [111Cd is expected to be collected along with 111Ag [In this context, the Legnaro National Laboratories of the Italian Institute of Nuclear Physics prompted a line of research aimed at the production of  project ,40,41. G project ,41. Acco(200 μA) ,41. Indeth 111Ag ,38,41, bth 111Ag .103Ag, 104m,gAg and 111Ag) have been revised and discussed. While the production of the dual therapeutic and SPECT-diagnostic 111Ag has been extensively explored in the literature, its related PET-imaging counterparts, 103Ag and 104m,gAg, have still been scarcely considered. As a result, the production of highly specific activity 111Ag could be achieved by the neutron irradiation of a 110Pd-enriched target or, alternatively, by low-energy (around 5 MeV) deuteron-induced reactions on the same target. Across both, the first pathway must be preferred due to the higher yield per unit amount of target material. The production of pure 103Ag is still an unsolvable concern due to the certain co-production of 104m,gAg, whichever pathway is followed. That being stated, the highest yielding process appeared to be the proton irradiation of 104Pd-targets via several  reactions. Concerning 104m,gAg, it can be theoretically obtained in a low yield but sufficiently high molar activity by irradiating highly enriched 104Pd-targets with protons in an energy range from 5 to 15 MeV, where the formation of 103Ag is restricted. The independent production of 104mAg and 104gAg is, conversely, thus far inconceivable. An alternative approach to address the lack of sufficiently pure diagnostic isotopes of silver, could be the coupling of the therapeutic 111Ag with a diagnostic radionuclide of a different element with sufficiently similar chemical behavior, such as the case of the “improper” matching pair formed by gallium-68 (68Ga) and 177Lu. Belonging to the same chemical group of silver, the most suitable candidates might be the radioisotopes of copper . However, the chemical and coordination properties of Cu2+ (which is the species afforded for copper radionuclides) are quite different from those of Ag+ (which is the species afforded for silver radionuclides), so such a pairing is barely warranted.In the present review, several routes for the production of silver radioisotopes of potential medical interest  chloride extraction cannot be admissible either, as it likely introduces traces of impurities that would affect the medical safety of the final product. Despite their efficiency, other processes referred to in this section do not seem suitable for radiopharmaceutical production directed to medical applications either, as their automation appears challenging. As a result, an optimal separation process that allows the sourcing of radioactive silver in a formulation that can be directly used for the labeling of biological vectors , avoiding the tedious steps of evaporation and re-dissolution, has not been reported to date. The test labelling of chelators with small amounts of 111Ag has only been attained up to now [Regarding the chemical separation methods, sundry studies, mainly restricted to the p to now .103Ag, 104m,gAg and 111Ag are very attractive radioisotopes for personalized medicine, embedding therapeutic and diagnostic potential in a triplet of the same element, research in this field still has many open avenues for the scientific community. Demanding challenges entail reliable and available means of production, as well as proper purification methods able to provide 103Ag, 104m,gAg and 111Ag in ready-to-use formulations. Among the three radionuclides, 111Ag is hitherto the most studied, and several reports regarding its production and purification are already available. The scaling up of the production in order to reach amounts comparable to other radionuclides used for therapy—such as, for instance, 177Lu, that exhibits almost equivalent emission properties—appears to be feasible in affordable ways both by traditional methods  and with high-energy cyclotrons coupled with on-line mass separation. Due to its certain potential, 111Ag is one of the novel radionuclides available in the PRISMAP portfolio (https://www.prismap.eu/radionuclides/portfolio/111Ag/ accessed on 10 November 2022) and small amounts of purified radionuclides are possibly provided upon the submission of research projects of mutual interest, aiming to foster the development of this isotope. Moreover, the decay properties of 111Ag make it suitable for perturbed angular correlation (PAC) spectroscopy and its possible applications have recently increased the interest of researchers in the field [103Ag and 104m,gAg appears more challenging but, due to the similarity of the half-lives and of the β+-emissions in terms of energy and abundance, the mutual contamination of the two radionuclides in the same solution should not be a concern and might be potentially used for imaging purposes as well. Due to the shortage of knowledge related to stable Ag-complexes in physiological conditions, deeper chemical and radiolabeling studies, able to provide suitable chelators for preclinical and clinical applications, are mandatory in order to foster the widespread application of these silver radionuclides; however, notable efforts have already been attempted in providing sulfur-rich macrocyclic ligands able to coordinate 111Ag with good preliminary outcomes [Although outcomes .


Aim: The main aim of this study was to assess the correlation between serum tumor necrosis factor-alpha (TNF-α) levels the and clinical severity of tuberculosis.Methods: This was a hospital-based case-control prospective study and was conducted at the Sher-i-Kashmir Institute of Medical Sciences, a tertiary care hospital in the northern part of India, from May 2016 to May 2018. The subjects were recruited in the study considering inclusion and exclusion criteria. All patients with pulmonary tuberculosis as well as patients with extrapulmonary tuberculosis were included as subjects and a clinical severity score based on anemia, weight loss, presence of hypoxia, and radiological features was calculated and compared with TNF-α levels. Age- and sex-matched healthy individuals were recruited as controls.Results: A total of 75 subjects comprising 50 cases and 25 controls were taken for the study. There were 34 (68.0%) patients with elevated TNF-α levels while only 16 (32.0%) patients had normal TNF-α levels. And, TNF-α levels were normal in 21 (84%) control subjects as compared to tuberculosis (TB) patients. Such difference in serum TNF-α levels between cases and controls was statistically significant (p<0.05). The mean serum TNF-α level in TB cases was 1265.63 pg/mL, while the mean serum TNF-α level in controls was 312.06 pg/mL. The difference in serum TNF-α levels between the two groups was statistically significant (p<0.01). We observed a significant increase in serum TNF-α levels with the increase in clinical severity score.Conclusion: Serum TNF-α levels were significantly associated with increased severity of TB. Tuberculosis (TB) is a major cause of death around the world. The infected cells and tissue macrophages in tuberculosis infection release a significant amount of cytokines. Studies have indicated the key role of TNF-α in infection control, mycobacteria eradication, and the development of granulomas . In HIV-+ cells (CTLs {cytotoxic lymphocytes}) engage in cytotoxic actions [Mycobacterium tuberculosis antigens presented by MHC coupled to HLA-I (human leucocyte antigen-I) complex are recognized; and (3) infected cells are induced to undergo apoptosis [+ cells in the bronchoalveolar lavage (BAL) fluid of patients with pulmonary TB [M. tuberculosis six months earlier, TNF-α neutralization increased the lung bacterial load [M. tuberculosis in human monocytes, as well as the rise in serum TNF-α levels and the worsening of a patient's clinical condition in cases of severe TB, are related [The cell-mediated immune response is essential for TB resistance, mainly through the production of cytokines, such as interleukin-2 (IL-2), interferon-gamma (IFN-γ), interleukin-12 (IL-12), interleukin-18 (IL-18) and tumor necrosis factor-alpha (TNF-α). When glycolipid antigen molecules are delivered to T lymphocyte cells by major histocompatibility complex (MHC) class I proteins in conjunction with cluster of differentiation 1d (CD1d) glycoprotein molecules, the TCD8 actions . In tubepoptosis ,7. Becaupoptosis , the stionary TB , and theonary TB . Further related ,14. Inte related ,16. TherThis is a prospective study conducted at the Sher-i-Kashmir Institute of Medical Sciences in the Department of Internal Medicine from May 2016 to May 2018. The informed consent of all patients and healthy controls were obtained. This study investigated the relationship between serum TNF-α levels and the clinical severity of tuberculosis. Clinical data, including anemia, fever, weight loss, dyspnea, night sweats, cough, sputum, and systemic examination were analyzed for patients. Diagnostic tests, including purified protein derivative (PPD) skin testing, erythrocyte sedimentation rate and blood counts, thoracic x-rays, abdominal x-rays, CT/MRI brain, CT scans of the chest, and CT scans of the abdomen/CT enterography, were also performed, depending on the location of the disease. Acid-fast bacilli positivity in fluid, tissue, or culture in each case, or a high adenosine deaminase (ADA) , were used to diagnose tuberculosis.The results of the thoracic radiography, the computed tomographic (CT) scan of the chest, CT brain, MRI (magnetic resonance imaging) brain, CT scan abdomen, and PPD skin test were coded as 0 (negative) or 1 (positive). The clinical severity of each case was determined through the following score: without anemia=0, with mild anemia=1 (11 g/dL or more), with moderate anemia=2 (8-11 g/dL), with severe anemia=3 (≤8 g/dL); without weight loss=0, with weight loss=1; without hypoxia=0, with hypoxia=1; thoracic x-ray/CT chest normal=0, thoracic x-ray/CT chest altered without cavity=1, thoracic x-ray/CT chest altered with cavity=2.TNF-α assaySera were obtained from diagnosed pulmonary and extrapulmonary patients, and healthy individuals (controls). A highly sensitive diaclone enzyme-linked immunosorbent assay (ELISA) method was used for measuring TNF-α levels. The sensitivity or the minimum detectable dose of TNF-α using this high-sensitivity diaclone TNF-α ELISA kit was found to be 0.5 pg/mL. This was determined by adding three standard deviations to the mean OD obtained when the zero standards were assayed 35 times. However, we considered 1.0 pg/mL as the minimum value below which the interleukin levels were treated as undetectable in order to be in line with most of the moderately sensitive ELISA kits used in our state (northern part of India).Statistical analysisIBM SPSS Statistics version 27.0  for Windows was used for the statistical analysis. Continuous data were presented as descriptive statistics , whereas categorical variables were represented by frequencies and percentages. Also, the association was done by using the chi-square test and r-value calculation using Pearson's correlation, the correlations between each case's TNF-α level and all clinical and diagnostic variables, and all results were discussed at 5% level of significance, i.e., p<0.05.EthicsThe study was carried out in accordance with the institutional ethical committee's recommendations and the 2013 revision of the 1964 Helsinki Declaration. The Institutional Ethics Committee (IEC) of Sher-i-Kashmir Institute of Medical Sciences (SKIMS) gave its approval to this study.Seventy-five (n=75) subjects including 50 cases and 25 controls were recruited for the study. Almost all the cases attended the hospital with a clinical presentation of TB. In this study, 54% (n=27) of the cases were males and 46% (n=23) were females with male to female ratio of 1:0.85. Based on age, the patients were grouped into two categories, less than 40 years and greater than 40 years of age. The number of cases in the age group of >40 years  exceeded those <40 years . The mean age of the patients was 43.5±13.9 years. Based on the smoking status, 20 (40%) patients were smokers, 16 (32%) were non-smokers and 14 (28%) were passive smokers. The differences in smoking among cases and controls were statistically significant (p<0.05). The subjects were considered non-smokers only if up to the day of sample collection they had not used tobacco and current smokers if they are smoking presently or had quit smoking in the last <6 months before sample collection. There was almost a similar representation of cases from rural and urban areas. There were overall no significant differences between cases and controls among different parameters like weight, socioeconomic status, and TB-associated co-morbidities (p>0.05). However, positive history of TB among subjects was significantly associated with cases (p<0.05) (Table There were 34 (68%) patients with elevated TNF-α levels while only 16 (32%) patients had normal TNF-α levels, the difference was statistically significant (p<0.05) (Table There were 34 (68%) patients with elevated TNF-α levels while only 16 (32%) patients had normal TNF-α levels. While TNF-α levels were majorly normal in control subjects 21 (84%) as compared to TB cases. Such differences in serum TNF-α levels between cases and controls were statistically significant (p<0.05)  among cases and controls with respect to all the subgroups of age, gender, place of residence, socioeconomic status, and other associated co-morbidities. The presence of smoking among cases and controls was significant with elevated serum TNF-α levels (p<0.05). Similarly, a history of TB among blood relatives in combination with increased TNF-α serum levels showed a significant association with TB Table .M. tuberculosis stimulation of tissues and inflammatory cells activates monocyte-macrophages to generate this cytokine [In the current study, a total of 50 TB patients were considered for analysis with varied clinical manifestations of TB. We attempted to demonstrate that TNF-α could potentially act as a cytotoxic agent, especially when increased blood levels were observed. Cytokine escape into the serum is the cause of TNF-α being present in peripheral blood circulation. cytokine .The details of the patient groups displayed, highlight that TNF-α plays a key role in the various clinical manifestations of TB. The control group and the TB group had significantly different serum TNF-α levels. We found that the clinical severity of the disease and serum TNF-α levels were positively correlated when the cases were categorized as per clinical severity scores ,19. The However, in some cases, serum TNF-α levels were very low. We hypothesize that the immunological response to the infectious pathogen may have been quite subpar in these patients. Although some individuals had severe lymphadenopathy and necrosis that could be seen using imaging techniques, some of them had normal serum TNF-α values. In some patients with lymph node TB, we hypothesize that the immune response may be compartmentalized. The extent of pulmonary tissue damage was higher in those with the highest serum TNF-α levels. We speculate that TNF-α may directly contribute to this behavior.We measured blood TNF-α levels sequentially in a small number of patients and found that, with a decrease in serum TNF-α levels, the patient's physical condition invariably improved. On the other hand, a poor clinical course was observed when serum TNF-α levels did not drop throughout the evolution. These findings suggest that the serum TNF-α levels may be a reliable indicator of the clinical course of TB patients. After demonstrating that declines in sputum IFN-γ, TNF-α, and IL-8 closely follow and even precede mycobacterial clearance, Ribeiro-Rodrigues et al. concluded that these cytokines may be reliable markers of disease activity and inflammation in TB .Additionally, we observed a positive association between serum TNF-α levels and weight loss, abnormal chest x-rays, and positive PPD skin tests; similar findings were observed by Andrade et al. . Other sWe observed that those with abnormal chest x-rays had higher serum TNF-α levels, Andrade et al. reported comparable findings . Tsao etPatients who had smoked in various ways demonstrated a substantial link with TB, and the study's significant finding was the presence of TB in blood relatives. Smoke is one of the allergens that has been found to alter IgE repertoires in the nasal mucosa. Additionally, the stimulation of the allergen in the mucosa may cause a sequential switch from IgG or IgA to IgE. Also, it has been found that mucosal locations produce almost all of the serum IgE, and nasal mucosa IgE-secreting B cell and plasma cell populations see a considerably larger rise than peripheral cell populations. Although there are data on the heritability of TB, the specific mechanism of genetic regulation has remained elusive ,27. NumeOne of the objectives of our study was to demonstrate the relationship between high serum TNF-α levels and extremely severe clinical TB with considerable tissue damage. On the other hand, we saw that patient clinical improvement was accompanied by a decline in the serum cytokine level. For instance, when the cytokine serum level is increased for an extended period, we hypothesize that lowering high TNF-α serum levels may reduce tissue damage.LimitationThe study's limitation is the paucity of knowledge that exists currently about TNF-α and TB in our group. However, selection or recall bias and the relatively smaller sample size could be the study's weak points. This bias is reduced by using the same hospital setting.M. tuberculosis elimination is the main focus of TB treatment. We recommend that TNF-α immune modulation be examined in M. tuberculosis therapy in future investigations, particularly in instances with extremely high TNF-α serum levels.Future standard TB clinical evaluations should incorporate TNF-α periodic measures using bioassay or ELISA technologies as they may help in understanding the consequences of tuberculosis. More research, nevertheless, is required before drawing firm conclusions. Today,