Fabry disease (FD) is a rare X-linked, lysosomal storage disorder caused by mutations in the alpha-galactosidase gene and characterized by neurological, cutaneous, renal, cardiovascular, cochleo-vestibular and ocular manifestations. The aim of this study is to characterize morphological, functional and autophagy-lysosome pathway alterations of the ocular surface in FD patients.Eleven subjects with a diagnosis of FD and fifteen healthy control subjects were examined. All patients underwent ocular surface slit lamp examination, corneal aesthesiometry and in vivo confocal laser-scanning microscopy (CCM). Conjunctival impression cytology was performed in six FD patients and six controls, to assess for expression of two markers of the autophagy-lysosome pathway: the microtubule-associated protein light chain 3 (LC3) and lysosome-associated membrane protein 2 (LAMP2).p < 0.001). No changes were observed in the conjunctival expression of LAMP2 between the two groups.Cornea verticillata and increased conjunctival vessel tortuosity were detected respectively in 67% and 33% of patients with FD. Compared with healthy subjects, patients affected by FD showed a significant reduction in corneal nerve fiber length, density and nerve branching on CCM and a significantly increased expression of LC3 on conjunctival impression cytology (This study shows that FD is associated with ocular surface alterations including corneal and conjunctival morphology, innervation and vascularization changes. Our data demonstrate an increased expression of LC3 protein in patients with FD, suggesting that alteration of the autophagy-lysosome pathway may play a role in the occurrence of ocular manifestations. Fabry disease (FD) is a rare, X-linked, lysosomal storage disorder characterized by total or partial functional deficiency of the enzyme α-galactosidase A, caused by mutations in the α-galactosidase A gene (GLA). A reduction or absence of activity of this enzyme determines an altered metabolism of glycosphingolipids, such as Globotriaosylceramide Gb3) –3 and ga –3 and gThe estimated incidence in the United States is between 1:40,000 and 1:117,000 live births for males , 8, 9.Ocular manifestations are observed in most FD patients, and involve the cornea, conjunctiva, lens and retina –13. The Currently there is on average a 15-year delay in the diagnosis of FD, due to the lack of awareness about the disease and non-specific symptoms , 24. TheChung et al. observed that the altered autophagy typically found in this disease could play a role in renal tubulointerstitial fibrosis and suggest that this pathway should be studied as a new therapeutic target in FD . This stEleven patients with clinical and biochemical diagnosis of FD, confirmed by genetic tests showing mutations on the GLA gene were included in the study . In addition, 15 healthy, sex and age matched subjects were included as a control group. Patients who were contact lens users, and/or who were affected by ocular or systemic diseases which may influence clinical and biological examinations, and/or who were in treatment with topical ophthalmic drugs were excluded from the study.All subjects underwent the following ophthalmological examination: (i) slit lamp biomicroscopy to detect anterior segment abnormalities including the presence of cornea verticillata and conjunctival vessels tortuosity, (ii) assessment of corneal sensitivity by Cochet-Bonnet aesthesiometer , and  2 proteins.All subjects underwent examination with a CCM . One eye of each FD patient and healthy subject, was randomly selected for examination and anesthetized with one drop of oxybuprocaine hydrochloride 0.4%. A large drop of gel was applied to the tip of the lens, and another small amount of gel was applied to the front surface of the Tomocap; the instrument was aligned with the center of the cornea, and advanced forward until it touched the cornea.−1); (2) the corneal nerve fiber density (NFD), measured as the total number of major nerve fibers per scan area (mm−2); and (3) the corneal nerve branch density (NBD), calculated as the number of branches emanating from all major nerve trunks per scan area (mm−2).The entire corneal epithelium was scanned in order to find an increased reflectivity of the layers, suggesting the presence of Gb3 deposits; going even deeper, the subepithelial corneal nerve plexus was evaluated. The images obtained were evaluated by a masked operator (P.M.) who selected the most representative images of the plexus of each subject and evaluated using NeuronJ (ImageScience): (1) the corneal nerve fiber length (NFL), calculated as the total length of all nerve fibers and branches per scan area , and the endogenous peroxidase block was performed by incubating the cells for 3 min with 3% hydrogen peroxide. They were subsequently incubated for one hour at room temperature with goat serum (NGS) diluted in 10% PBS, and finally incubated overnight at 4 °C with the primary antibodies . The following day, they were incubated sequentially with a secondary anti-rabbit antibody conjugated to horseradish peroxidase (HRP) and with a streptavidin / peroxidase complex . The immune complexes were visualized through ImmPACT DAB (Vector). The electronic images were acquired through bright field microscopy and collected in CS5 Adobe Photoshop format. The densitometries related to the immunopositivity of the photographed fields were made through the ImageJ Software for Windows program.P-value < 0.05 was considered statistically significant.Quantitative variables were expressed as mean and standard deviation. The qualitative variables were expressed as percentages. To verify the existence of statistically significant differences in measured values of corneal sensitivity, corneal nerve morphology parameters and LC3 and LAMP2 protein expression between patients with FD and healthy subjects, an independent samples t-test was used. Spearman-rho test was used to evaluate the correlation between the variation of nerve morphology parameters evaluated and corneal sensitivity values. p = 0.0325).Eight patients (70%) with FD showed cornea verticillata. Four patients (35%) with FD showed increased conjunctival vessels tortuosity in the inferior part of bulbar conjunctiva at slit lamp examination. Fig. . Patientp = 0.040; NFD: FD: 25 ± 10.46 vs healthy controls 52.78 ± 20,98 fibers/mm2, p = 0.001; NBD: FD: 13,07 ± 8.59 vs healthy controls 40,28 ± 17,43 branches/mm2, p < 0.001) , changes of corneal nerve morphology were significantly correlated with the decrease of corneal sensitivity: NFD , NBD .Although corneal sensitivity values measured by Cochet Bonnet aesthesiometer did not significantly correlate with NFL values (p < 0.001). No statistically significant difference in the expression of LAMP2 was detected  and increased of conjunctival vessels tortuosity (35%). Currently, cornea verticillata is considered the ocular hallmark of FD. The observation of an increased conjunctival vessels tortuosity, mainly located at the inferior part of bulbar conjunctiva, in 35% of FD patients is in line with previous data and related to the progressive accumulation of Gb3 in endothelial cells –21.Our data also showed that approximately 20% of FD patients had an increased reflectivity of the basal cells layer of corneal epithelium, as detected by CCM, suggesting a possible employment of this diagnostic technique to improve the identification of the Gb3 deposits , 16, 17.Immunohistochemistry performed on conjunctival impression cytology samples showed increased levels of LC3 protein in FD patients when compared with the control group.An increased expression of autophagosome LC3 protein, due to impaired autophagic function, is a common observation in lysosomal storage disorders, such as Fabry disease, as demonstrated by immunohistochemistry performed on renal biopsy samples . The autOur results did not show significant changes of conjunctival levels of LAMP2, although an increase in the expression of this protein in phagocytes of Fabry patients has been previously demonstrated . This coThis study highlights the marked alteration of the ocular surface in FD patients and suggests to further evaluate the clinical use of CCM and conjunctival impression cytology, two simple/non-invasive diagnostic techniques, to monitor Fabry disease progression and response to treatment.


Pseudomonas syringae pv. tomato is a serious foliar disease on tomato. However, it is still unknown how organic fertilizers application mediates plant defense against foliar pathogens by altering the composition of the soil microbial community. We conducted a 2-cycle pot experiment involving chemical and organic fertilizers and tracked tomato foliar pathogen incidence. Using microbiome sequencing, we then compared the differences in bulk and rhizosphere microbial communities. The results showed that, compared with soils amended with chemical fertilizer, soils amended with organic fertilizer gradually and significantly presented a reduction in tomato foliar disease, and the bacterial richness and diversity significantly increased. Moreover, the bacterial and fungal compositions of the bulk soil and rhizosphere soil of the organic fertilizer and chemical fertilizer treatments were different from each other. More importantly, the abundance of some potentially beneficial bacteria, such as Luteolibacter, Glycomyces, Flavobacterium, and Flavihumibacter, increased in the organic fertilizer-amended soil, and these genera were significantly negatively correlated with the incidence of tomato foliar disease. These results suggest that organic fertilizers can alter the taxonomy of the soil microbiome and that some specific beneficial microbial communities may play an important role in reducing the infection of foliar pathogens by inducing plant resistance.Bacterial speck caused by  Large amounts of chemical fertilizers (CFs) are applied to soils, causing various major threats, such as soil acidification, nitrogen leaching, a decrease in soil organic matter and an increase in plant diseases . ConsequArabidopsis thaliana specifically promotes Microbacterium, Stenotrophomonas and Xanthomonas spp. in the rhizosphere upon foliar defense activation by downy mildew-causing pathogens. Bacterial speck disease by Pseudomonas syringae pv. tomato is common disease throughout tomato growing areas. It has been found that P. syringae pv. tomato mainly infects plant leaves, causing plant tissue necrosis, and resulting in 75% of tomato yield loss. The influence of rhizosphere microorganisms on plant diseases has always been a popular but difficult topic in the field of plant pathology research both in China and in other countries. Nonetheless, how OF alters rhizosphere microbiota protect against aboveground plant pathogen attack remains unclear.In agricultural production, one method to overcome the occurrence of pests and diseases involves the improvement of soil properties by fumigation, crop rotation and OF application . Soil fuCF and OF treatments, and each treatment involved inoculation with P. syringae pv. tomato DC3000 (Pst). The experiment was carried out for 2 cycles, the disease incidence of each cycle was measured, and soil samples were collected to analyze bacterial and fungal community composition via high-throughput sequencing at the end of the second cycle. Reviewing both the relationship between OF application and the soil microbiome and the impact on foliar disease is important with respect to applying OFs and foliar disease prevention and control.In this study, the effect of OF on tomato foliar disease was examined. Experiments were divided into The soil used in the study was collected from a field with no history of tomato cultivation in Guangzhou, Guangdong Province, China , in March 2021. Topsoil samples (depth of 20 cm) were collected, thoroughly mixed and transported in plastic bags to South China Agricultural University, Guangzhou city, Guangdong Province, China. All the soil samples were dried at room temperature and passed through a 3-mm sieve to remove other plant roots and gravel. To restore the microbial community, tomato seedlings were transplanted and allowed to grow for 4 weeks in collected soil, after which the soils were sieved again to remove the plant material. The soil was stored at 4°C until the start of the experiment.−2 s−1, 70% humidity). Two different fertilizer treatments were applied—OF and CF. The pot experiment was performed in accordance with a randomized complete block design with 3 replicates for each treatment, each replicate involved 4 pots, and each pot (15 cm diameter and 13 cm height) was filled with 2 kg of soil. OF (chicken manure and mushroom dreg compost) was supplied by LeFu Co., Ltd., Huizhou city, Guangdong Province, China. The test treatment was amended with 40 g of OF and the control treatment was amended with 0.51 g N (urea), 0.44 g P (superphosphate), 0.40 g K (potassium chloride) before each cycle.A pot experiment was carried out in a climate chamber  was inoculated into the wounds. After the Pst suspension was allowed to dry naturally on the leaves, pots were randomly placed back into the growth chamber and covered to increase the humidity. Each pot was watered every 2–3 d to maintain the soil moisture content between 60 and 80% of field capacity. Subsequently, all the plants were removed. A 2-cycle tomato plantings were carried out as described above, with the same procedures applied to each cycle. Each successive cycle used soil from the previous cycle after plant removal. Moreover, in the second cycle, the plants received the same concentrations of OF and CF as that applied in the first cycle before planting.Tomato seeds were surface-sterilized with 3% sodium hypochlorite for 3 min followed by 70% ethanol for 1 min and then germinated on water–agar plates for 2 d. The seeds were sown onto seedling plates (each well contained 60 g of seedling substrate). At the three-leaf stage, three tomato plants were transplanted into each pot. At 10 d after transplantation, all the leaves of tomato plants were punctured, and 2 μl of Pst. The typical symptoms caused by Pst were the leaves show dark brown specks or black specks that necrotize quickly. Therefore, we defined diseased plants based on the typical bacterial speck disease symptoms observed. The disease incidence was calculated by counting the number of tomato plants with bacterial speck disease symptoms among the total number of plants.In each cycle, the disease incidence of tomato leaves was recorded 21 d after inoculation with ® SPIN Kit for Soil  following the manufacturer’s instructions. Subsequently, the concentration and quality of the DNA samples were determined by a spectrophotometer .Soil sampling was performed at the end of the second cycle. In brief, 5 healthy tomato plants were randomly chosen in each treatment, and the bulk and rhizosphere soil samples were obtained according to the methods of ® High-Fidelity PCR Master Mix (New England Biolabs), each primer at 0.2 μm and 10 ng of target DNA. The cycling conditions consisted of an initial denaturation step at 98°C for 1 min, followed by 30 cycles of 98 (10), 50 (30), and 72°C (30 s) and a final 5 min extension at 72°C. The PCR products were mixed in equal proportions, and then, a Qiagen Gel Extraction Kit  was used to purify the mixed PCR products. The amplicons were sequenced using an Illumina NovaSeq PE 250 platform at Novogene Co., Ltd., Beijing, China.Amplification of bacterial 16S rRNA within the V3-V4 regions was amplified from the soil DNA with the specific primers 341F—806R and a barcode was attached to the forward primer, while the fungal-specific primers ITS1F—ITS2 were used to target the fungal ITS2 region. All the PCR mixtures included 15 μl of PhusionPaired-end reads were assigned to samples based on their unique barcodes and were truncated by cutting off the barcodes and primer sequences. Paired-end reads were merged using FLASH (Version 1.2.11),The bacterial and fungal diversity were analyzed using the Shannon index, and the Chao 1 index was calculated to evaluate the community richness of the samples. Principal coordinate analysis (PCoA) based on a Bray–Curtis dissimilarity matrix was performed to visualize differences of the samples in complex multidimensional data, and coordinates were used to draw 2D graphical outputs. Analysis of molecular variance (AMOVA) was used to detect the significant differences in microbial communities between the different fertilization management treatments. Afterward, linear discriminant analysis (LDA) of effect size (LEfSe) was performed to determine the significantly abundant bacterial and fungal taxa within the two treatments. Moreover, Spearman rank correlations between the relative abundance of the rhizosphere genera and the disease incidence were used to analyze potentially beneficial microbial genera in the rhizosphere. A co-occurrence network analysis was subsequently performed on the rhizosphere microbiome of each treatment to explore the interactions between ASVs. Additionally, PICRUSt software was used to predict the bacterial metabolic functions by the use of information within the Kyoto Encyclopedia of Genes and Genomes (KEGG) database .p < 0.05. Disease incidence was calculated by the percentage of diseased tomato plants out of the total number of tomato plants in each replicate.Statistical analyses were performed by using IBM SPSS 20.0 software , and statistical tests performed in this study were considered significant at Pst, but the disease incidences were not significantly different  between CF and OF  reduced disease symptoms compared to the those of seedlings grown in the CF treatment , organic matter (OM) content, and available potassium (AK) content. Moreover, Mantel tests showed that soil physiochemical properties were not significantly associated with disease incidence .The successive plant experiments showed that OF treatment reduced the incidence of foliar disease in tomato seedlings . In cyclF and OF . When threatment . In addireatment . Comparevia 16S rRNA gene and ITS2 region amplification, respectively. In total, 1,688,451 and 1,511,186 high-quality sequences were obtained from bacteria and fungi, respectively. The average read counts per sample containing bacteria and fungi were 75,559  and 84,423 , respectively. Unlike for fungi, for bacteria, significantly  higher bacterial diversity (Shannon) and richness (Chao 1) were observed in organic fertilizer-amended bulk (OFB) soil and rhizosphere soil (OFR). However, the fungal diversity (Shannon) was observed to be higher in the chemical fertilizer-amended bulk (CFB) soil and rhizosphere soil (CFR), but no significant value of fungal richness (Chao 1) was observed between the two different fertilization management programs  and fungal  community compositions under the different fertilization management practices along the first principal component (p < 0.01) from those in the rhizosphere along the second principal component.The diversity of bacteria and fungi under the different fertilization management programs was analyzed programs . To visuomponent , the comr = −0.70, p = 0.03) and diversity  were negatively correlated with disease incidence, while no significant correlations were observed for fungal richness  or diversity  and fungal  community compositions were significantly correlated with disease incidence. Furthermore, Spearman rank correlation analysis was performed between the relative abundance of sensitive bacterial and fungal genera and disease incidence. A total of 35 sensitive bacterial genera and 1 sensitive fungal genus showed a significant negative correlation with disease incidence in the rhizosphere soil . Furthermore, the relationships between soil physicochemical properties and the sensitive bacteria were analyzed by Spearman rank correlation. As shown in 4+-N content, EC, and OM content were significantly positively correlated with the most sensitive microbial genera.Considering the importance of the rhizosphere microbial community composition in plant disease resistance, Mantel tests were performed to determine the correlations between bacterial and fungal community composition and disease incidence. The results showed that the rhizosphere bacterial (ere soil . The difere soil . Some ge the CFR . In the  the CFB . In addi and CFR . Interes the OFR . Other svia KEGG), PICRUSt was used to predict the function of the 16S rRNA gene sequence. The tomato plant rhizosphere bacterial community that developed in response to the different fertilizer treatments were more similar in terms of function than taxonomic classification  and fungal  compositions were significantly correlated with disease incidence, which is in agreement with the results of a previous study . Previous studies have reported that pH, EC, and OM content are generally negatively correlated with soil-borne disease severity  can be found at: XL and YY conceived and designed the study. FH conducted experiments and wrote the manuscript. LL, CM, and JS analyzed the data. All authors contributed to the article and approved the submitted version.This work was supported by the Modern Agro-Industry Technology Research System (CARS-40) and the Science and Technology Program of Guangdong province, China (2020B1212060060).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study therefore presents the cardiovascular risk factors and the underlying aetiology of HF among admitted patients in a teaching Hospital in Ghana.Heart failure (HF) is recognized as a global public health disease associated with high morbidity and mortality. It is suggested that the main underlying causes of HF in developing countries differ from those identified in well-resourced countriesThe study prospectively recruited 140 consecutive patients admitted for heart failure at the Medical department of the Korle-Bu Teaching Hospital from March to October, 2014. The study evaluated the cardiovascular risk factors and the aetiologies of heart failure, and compared the risk factors and aetiologies with patient’s age and gender.p = 0.004) and those presenting with multiple risk factors (p = 0.001).The mean age of the study participants was 51.3 ± 16.8 years. The commonest cardiovascular risk factors observed were hypertension (46.5%), history of previous HF (40.7%), excessive alcohol use (38.6%), and family history of heart disease (29.3%); predominantly hypertension (68.3%). The major underlying aetiology of HF were dilated cardiomyopathy (38.6%), hypertensive heart disease (21.4%), ischaemic heart disease (13.6%) and valvular heart disease (12.9%). These underlying aetiology of HF were more common in patients aged 40 years and above (The major underlying aetiology of heart failure in adults were dilated cardiomyopathy, hypertensive heart disease, ischaemic heart disease and valvular heart disease, which were significantly high among patients aged 40 years and above and those presenting multiple risk factors. Hypertension, excessive alcohol use, family history of heart disease and personal history of previous heart failure diagnosis are noted as the main cardiovascular risk factors among heart failure patients. Heart failure (HF) has been singled out as an emerging epidemic –3. In deHF is a common terminal condition among many common chronic cardiovascular diseases in Ghana and accounted for about 8% of all admissions  and alsoThis was a hospital-based cross-sectional case study recruiting every consecutive patient who was admitted for acute heart failure (AHF) during the study period at the Department of Medicine & Therapeutics (DOMT) of the Korle-Bu Teaching Hospital (KBTH). KBTH is a tertiary referral center located in the national capital of Ghana, Accra with 1600 bed capacity and twelve different departments. The DOMT is a 134-bed facility operating under four teams and has a patient flow of about 300 per month. The department runs a 24-h emergency service and admits patients through the emergency department, outpatient clinics and other specialist clinics. Referrals are received mainly from the southern sector of Ghana but also from all the 16 regional hospitals, district hospitals, private hospitals and specialist clinics. Other referrals are received from polyclinics, maternity homes and some health centers.This study involved all adult patients who were admitted and managed for AHF in all four medical wards and the emergency department of the Korle-Bu Teaching Hospital from March to October 2014. Consecutive patients 18 years and above who were admitted with initial diagnosis of AHF and who met the modified Framingham criteria  for the Paroxysmal nocturnal dyspnoea, neck vein distention, pulmonary rales, radiographic cardiomegaly (cardiothoracic ratio > 0.50), acute pulmonary oedema, S3 gallop, and hepatojugular reflux.Bilateral ankle oedema, nocturnal cough, dyspnoea on ordinary exertion, hepatomegaly, pleural effusion, tachycardia (heart rate > 120/min) and weight loss of more than 4.5 kg in 5 days in response to treatment of congestive HF . Minor c.Patients who did not meet the Framingham criteria, were unable to complete full diagnostic evaluation, were pregnant, or were less than 18 years were excluded from the studyThe sample size for the study was determined using the formula for single cross-sectional survey based on the parameter; earlier studies in Ghana, the expected prevalence (p) of AHF among medical admissions of 8% , the sta0inclinations unless the level of dyspnoea did not permit this position, in which case the examination was done in a sitting position. Physical examination was done with emphasis on looking for signs of HF and pointers to probable causes and risk factors. Physical examination assessed the Heart rate, pulse character, jugular venous pressure, Body mass index (BMI), blood pressure (BP), presence of third heart sound (S3), Hepatomegaly, Hepatojugular reflux, bilateral basal crepitations and pitting ankle oedema.A standard questionnaire which was administered by the principal investigator (a cardiologist) and a trained research assistant (a physician) was used to collect data including demographic characteristics , presenting complaints, and symptoms of HF, history of hypertension, diabetes, dyslipidaemia, smoking history, drug history and previous diagnosis of HF, and family history of heart diseases after informed consent was obtained. Clinical examination was done by the first author (a cardiologist), guided by modified Framingham criteria  and the A standard postero-anterior Chest X-ray was done by a certified radiology technologist, and examined by the first author for cardiomegaly, pulmonary oedema , pleural effusion, consolidation, fibrosis and other lung diseases. Echocardiographic and electrocardiographic data were collected within five days of admission. All the patients had 2D, M-mode, and Doppler Transthoracic echocardiography (TTE) as part of a comprehensive evaluation done by the first author (a cardiologist) with expert guidance from supervisors (experienced cardiologists) and in accordance with standard guidelines, e.g., , 19 usinData collected were entered, cleaned and analyzed using SPSS version 20 software. Descriptive analysis of the cardiovascular risk factors and underlying aetiology of heart failure was done. Continuous variables were summarized using mean and measure of spread using standard deviation and range were also calculated. Chi-square and Fischer exact tests where appropriate were done to compare the prevalence of cardiovascular risk factors and major underlying aetiologies of HF by gender and age.The mean age of the study participants was 51.3 ± 16.8 years. There were more males (56.4%) than females (43.6%). Patients were mostly urban settlers (78.6%). The socio-demographic characteristics of the patients are presented in Table Table p = 0.001), diabetes  and multiple risk of 3 or more factors  were significantly prevalent among patients aged 40 years and above compared to those aged below 40 years. Male patients compared to female patients had high prevalence of smoking , and alcohol use . However, more females than males  had BMI of 30 kg/m2 and above.The history of hypertension  and presenting with multiple risk of 2 or more factors (p < 0.001) were diagnosed more with hypertensive heart disease, dilated cardiomyopathy ischaemic heart disease and valvular heart disease compared to those aged less than 40 years and presented with one or no risk factor respectively.Table The aim of the study was to examine the prevalence of cardiovascular risk factors and underlying aetiology of acute heart failure at the Korle-Bu Teaching Hospital, the national referral center serving the southern sector of Ghana.2) was significantly higher in females and those aged 40 years and above. This is consistent with the findings made in a previous study where the history of former or current smoking was frequent among male patients and female patients were more obese [The study found that hypertension was the most common risk factor for AHF. This is consistent with several studies done in Ghana , 14, 21 re obese . The curre obese . Cardiovre obese , which mThe study identified cardiomyopathies and hypertensive heart disease as the most common underlying aetiology of HF in this study. Other common underlying aetiology were ischaemic and valvular heart diseases. This is similar to the findings of the studies by Owusu et al. in Komfo Anokye Teaching Hospital in Kumasi , 15, andHypertensive heart disease results from chronic systemic arterial hypertension and has emerged as the commonest cause of AHF in several studies in Africa , 22, 28.Furthermore, ischaemic heart disease was found as more common than primary valvular heart disease in the aetiology of HF. This finding is at variance with the findings in earlier studies in Ghana , 15, theOur current study found only 12.9% of the AHF was due to primary valvular heart disease. Moreover, most of the valvular heart diseases were degenerative rather than rheumatic, reflecting the ageing population. A similar low prevalence of rheumatic heart disease as a cause of contemporary HF was found in Abeokuta, Nigeria in a recent registry in which only 2.4% of the 452 HF patients had rheumatic heart disease . Both stCongenital heart diseases were very few in this study, like that found in the Abeokuta HF registry where only 2 congenital heart disease cases were identified in the 452 adults with HF . This maThe study found that hypertensive heart disease, cardiomyopathies, ischaemic and valvular heart diseases were predominant in patients aged 40 years and above and those presenting with multiple risk factors. This is consistent with the finding made in a similar study among heart failure patients in a tertiary hospital in Kumasi, Ghana . It has A major limitation of this study is the inability to do natriuretic peptides on admission. This test was not routinely available in the facility during the time of the study. Diagnosis of ischaemic cardiomyopathy was based on history, electrocardiography, and echocardiography with no coronary angiography. Additionally, virological and serological tests were not done to investigate the underlying cause of dilated cardiomyopathies. A previous history of heart failure was established mainly by clinical history and may suffer from recall bias. The definition of hypertensive heart disease used for this study might have missed out patients with end-stage hypertensive heart disease with thinned walls.The study has shown clearly that the major underlying aetiologies of acute heart failure in adults admitted to Korle-Bu Teaching Hospital were dilated cardiomyopathy, hypertensive heart disease, ischaemic heart disease and valvular heart disease irrespective of gender. Dilated cardiomyopathy and hypertensive heart disease were the most common aetiologic factors, especially in patients aged 40 years and above and those presenting with multiple risk factors. Whilst efforts at controlling rheumatic heart disease and other infections should continue, equally greater policy attention should be given to non-communicable diseases like hypertension, diabetes, and coronary artery disease to reduce heart failure burden in Ghana.


Congenital central hypoventilation syndrome (CCHS) is a rare neurological genetic disorder that affects sleep-related respiratory control. Currently, no drug therapy is available. In light of this, there is a need for lifelong ventilation support, at least during sleep, for these patients. The pathogenesis of several chronic diseases is influenced by oxidative stress. Thus, determining oxidative stress in CCHS may indicate further disorders in the course of this rare genetic disease. Liquid biopsies are widely used to assess circulating biomarkers of oxidative stress. In this study, ferric reducing ability of plasma, thiobarbituric acid-reactive substances, advanced oxidation protein products (AOPPs), and advanced glycation end-products were measured in the serum of CCHS patients to investigate the relationship between oxidative stress and CCHS and the significance of this balance in CCHS. Here, AOPPs were found to be the most relevant serum biomarker to monitor oxidative stress in CCHS patients. According to this communication, CCHS patients may suffer from other chronic pathophysiological processes because of the persistent levels of AOPPs. The development and the progression of several chronic diseases are often associated with oxidative stress (OS), a phenomenon defined as an imbalance between the formation of reactive oxygen species (ROS) and antioxidant defense systems ,2. ROS aAdvanced oxidation protein products (AOPPs), derived from the action of hypochlorous acid and chloramine produced by neutrophil myeloperoxidase, are regarded as both oxidative stress and inflammatory biomarkers in clinical samples . Lipid pAdvanced glycation end-products (AGEs) are heterogeneous compounds generated from the nonenzymatic glycation of macromolecules  through the Maillard reaction; despite being often associated with hyperglycemia, increased AGEs have also been documented in normoglycemic subjects suffering from other conditions characterized by increased OS such as inflammatory diseases and obstructive sleep apnea ,7.Physiological homeostasis is maintained by sophisticated enzymatic and nonenzymatic antioxidant defenses that counteract excessive ROS levels . The FRAPHOX2B) gene is present in 90% of CCHS patients [PHOX2B is a key gene for the neuronal formation and differentiation, especially in the autonomic nervous system. Indeed, CCHS patients manifest episodes of apnea/hypopnea during sleep and during routine activities of daily living. This pathological condition results from abnormal control of breathing with reduced sensitivity to hypercapnia and hypoxemia [Congenital central hypoventilation syndrome (CCHS) is a rare autosomal dominant genetic disorder (ORPHA:661) that affects respiratory control , whereas determination of ROS levels requires very rapid sample processing (within 2 h from collection). In light of these considerations, we recently performed an HPLC–MS/MS analysis to detect the three major oxidative biomarkers, i.e., n minors .According to the assumption that OS contributes to the onset of chronic-degenerative diseases ,22, the Therefore, in this study, circulating antioxidant/oxidant status was evaluated by measuring FRAP, TBARS, AOPP, and AGEs in CCHS patients to evaluate the relationship between OS and CCHS, and to investigate the significance of this balance in this rare genetic disease.PHOX2B gene mutation were enrolled. Enrolled subjects received detailed explanations of the study, and all subjects signed a written, informed consent form; for minors, a parental written informed consent form was signed. The Institutional Review Board of Meyer Children’s Hospital  approved the study protocol (Ethics Committee registration number 99/2017).For this study, four minor healthy controls, five adult healthy controls, and nine CCHS patients (five minors and four adults) with confirmed All subjects were Italian and of the same ethnicity; they had no differences in height, weight, body mass index (BMI), and lipid profiles. Healthy controls were age-matched subjects without diseases, not taking drugs or alcohol, and nonsmokers. All CCHS patients had no additional phenotypic features of CCHS . ROS production is influenced by daily activities or individual-dependent factors . CCHS pag, 15 min). Serum samples were stored at −20 °C within 2 h of collection.All blood samples were collected fasting in the morning; for CCHS patients, blood was sampled after the polysomnography analysis. Blood was collected in nonadditive serum preparation tubes (red top). The serum tubes were kept at room temperature for 60 min before centrifugation (1000× v/v), and 160 µL of the resulting supernatant was added to 32 µL of thiobarbituric acid (0.12 M)  in 0.26 M Tris, before heating at 100 °C for 15 min. A 10 min ice bath and centrifugation  stopped the reaction, and the absorbance of the supernatant was measured at 532 nm .TBARS are markers of lipid peroxidation, and they were determined as reported in our previous work . Briefly−5 M·cm−1) was used to calculate TBARS content. TBARS were expressed as pmol/mg protein.The molar absorption coefficient  was diluted in 1 mL of PBS, and then mixed with 50 µL of KI (1.16 M) and 100 µL of acetic acid. The absorbance value was immediately read at 340 nm. Chloramine-T , as the standard for the calibration curve, was used to quantify AOPPs. AOPPs were expressed as nmol of chloramine per mg protein .According to Cournot and Burillo , AGEs we3·6H2O (10:1:1)) was freshly prepared incubated for 30 min at 37 °C. The ferric–tripyridyl–triazine complex is reduced into the ferrous form, developing an intense blue color. The absorbance value was measured at 595 nm using the Wallac 1420 Victor 3 Multilabel Counter . FeSO4·7H2O was used for the standard curve, FRAP was expressed as nmol/mg protein. Merck KGaA  supplied all reagents.Assays based on Benzie and Strain’s method were used to determine FRAP . As prevp-value < 0.05 was considered statistically significant. Normality was verified with the Kolmogorov–Smirnov test. Comparison of continuous variables between the two groups was performed using the Student’s t-test or Mann–Whitney test as applicable according to data distribution.Statistical analyses were performed using Graph-Pad Prism 7.00. A Oxidative Damage and Antioxidant Capacity in the Serum of CCHS PatientsBiomarkers of systemic OS and total antioxidant capacity are of great value in assessing a physiologically altered or increased disease state .p < 0.001). This finding is consistent with the elevated ROS levels we previously detected in blood cells of CCHS patients [As shown in patients . On the Since we recently demonstrated increased urinary MDA levels in young adult CCHS compared with age-matched control subjects , in thisp < 0.05) and adult CCHS patients (mean age 25.25 ± 2.13 years) compared to adult healthy controls (mean age 26.60 ± 1.28 years) .As depicted in AOPPs resulting from myeloperoxidase activation and hypochlorous acid production during inflammation are capable of binding to pattern recognition receptors (PRRs) whose downstream effects result in the activation of OS and inflammatory pathways including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor-κB (NF-κB) activation . Among t2+/mg vs. 8.17 ± 1.25 nmol Fe2+/mg, p < 0.05) whereas no difference was observed when comparing FRAP levels from adult CCHS to adult controls . Reports on FRAP values in healthy children and young adults are lacking, and data on antioxidants plasma levels are contradictory. The concentration of total thiols in the plasma of females, but not of males, progressively decreased in the group aged 20–39 compared to groups aged 6–13 and 14–19 [In addition, FRAP values were significantly lower in CCHS aged below 18 years compared to healthy minors , but most probably by the detection method; in fact, spectrophotometric TBARS determination is nonspecific for MDA since the thiobarbituric acid test measures reactive substances other than MDA, such as 2-alkenals and 2,4-alkadienals . It was PHOX2B mutations in this disease.Furthermore, this study showed that AGEs levels were not altered between patients with CCHS and healthy controls but were increased in healthy adults compared with healthy minors D. This eOverall, our results suggest increased OS in CCHS patients and that AOPPs are the most relevant serum biomarker for monitoring OS in these patients. It is noteworthy that AOPPs can cause cellular dysfunction, cell death, and tissue damage in different organs, thus representing promising biomarkers of the redox imbalance associated with CCHS and its complications.PHOX2B mutations, although some studies have suggested that transcriptional dysregulation and protein misfolding may underlie pathogenesis [In this study, the enrolled patients did not have hypoventilation during wakefulness; the apnea index and dyspnea index, oxygen desaturation index, and carbon dioxide levels in these patients were in the normal range during night ventilation. Therefore, we can rule out an association between the results obtained in this study and the possible ineffectiveness of artificial ventilation and sleep quality. However, to date, very little is known about the downstream cellular and molecular consequences of ogenesis ,40The biochemical mechanisms underlying rare diseases are generally poorly understood. In most cases, the chronic pathophysiological conditions associated with the disease, in addition to the patient’s clinical manifestations, are generally asymptomatic. However, they could be responsible for the onset of other chronic collateral disorders causing patients’ clinical worsening over the lifetime.Discoveries about molecules or signaling pathways associated with these latent pathophysiological processes could provide fundamental knowledge to intervene early against worsening clinical status.This communication adds new insights about the systemic oxidative state in CCHS patients and demonstrates for the first time that AOPPs are one of the key serum biomarkers of oxidation in CCHS.Because AOPPs are known to be stimulating factors of inflammatory responses, this study suggests a possible involvement of the inflammatory process in CCHS. In light of these considerations, further evaluation on the inflammatory state in these patients could contribute to the understanding of the pathophysiology of this rare genetic disease.Overall, the results obtained so far on CCHS generate hypotheses for future clinical trials aimed at improving the CCHS outcomes by hindering increased oxidative stress through the promotion of a healthy lifestyle, adequate diet, and the possible therapeutic use of antioxidant agents or pharmacological strategies targeting the AOPP/RAGE axis and activation of its downstream inflammatory pathways.


Appropriate fertilization can enhance forest productivity by maintaining soil fertility and improving the structure of the bacterial community. However, there is still uncertainty surrounding the effects of combined application of organic and inorganic fertilizers on soil nutrient status and bacterial community structure. A fertilization experiment was set up in an eight-year-old teak plantation with five treatments involved: mixed organic and NPK compound fertilizers (OCF), mixed organic and phosphorus fertilizers (OPF), mixed organic, NPK and phosphorus fertilizers (OCPF), mixed NPK and phosphorus fertilizers (CPF) and no fertilization (CK). Soil chemical properties and bacterial communities were investigated, and the co-occurrence pattern of the bacterial community under different fertilization treatments was compared. The results showed that the contents of soil organic matter and nitrate nitrogen, and the soil pH values were the highest after OCPF treatment, which were 20.39%, 90.91% and 8.16% higher than CK, respectively. The richness and diversity of bacteria underwent no obvious changes, but the structure of the soil’s bacterial community was significantly altered by fertilization. Of the dominant bacteria taxa, the relative abundance increased for Gemmatimonadetes, Myxococcota, ADurb.Bin063-13 and Candidatus_Koribacter, and decreased for Chloroflexi, Proteobacteria, JG30-KF-AS9 and Acidothermus under OCPF treatment in comparison to CK. The number of nodes and edges, the average degree and the network density of bacterial community co-occurrence networks were the greatest in OCPF treatment, indicating that application of OCPF could make the network structure of soil bacteria more stable and complex. Moreover, soil pH and organic matter were significantly correlated with bacterial community structure and were considered the main influencing factors. These findings highlight that the combined application of organic, NPK and phosphorus fertilizers is highly beneficial for improving soil quality and optimizing bacterial community structure in teak plantations. Fertilization is one of the pivotal management measures in the timber forest cultivation process. It directly affects soil fertility and forest productivity, as well as the diversity and composition of microorganisms by changing soil aggregate structure and nutrient levels ,2. ApplySoil microorganisms are an extremely abundant biotic group and are important for maintaining the diversity and stability of the terrestrial ecosystem ,16. BactBacterial communities are characterized not only by the composition and number of species, but also by the interactions and associations among different taxa . The bacTectona grandis L.f.) is one of the most precious hardwood species in the tropics, known for its excellent timber quality, and it is used extensively in the manufacture of high-grade furniture and wooden floors [Teak . This region has a subtropical monsoon humid climate with a mean annual temperature of 19.6 °C. The average annual rainfall is about 1400 mm. The daylight hours and annual frost-free period are 1350–1520 h and 335 days, respectively. The soil was identified as yellow−red earth and derived from sand shale. Teak seedlings of the fine clone 7544 were used for afforestation, which was received from the Research Institute of Tropical Forestry, Chinese Academy of Forestry in Guangzhou, China. The experimental plantation was planted in 2010 at a spacing of 2.5 m × 3.0 m.2 each were set up. Fertilization was conducted in May 2018 and 2019, and annual fertilizer inputs are listed in The fertilization experiment was established in a randomized block design and included five treatments: the combined application of organic and NPK compound fertilizers (OCF); the organic and CaMgP fertilizers (OPF); the mixed application of organic, NPK and CaMgP fertilizers (OCPF); the combined application of NPK and CaMgP fertilizers (CPF); and no fertilization as a control (CK). Each treatment was repeated four times. A total of 20 treatment plots of 270 m2O5 and K2SO4 in the ratio 14:16:15. CaMgP fertilizer contains 18% of P2O5, 45% of CaO and 12% of MgO. All inorganic and organic fertilizers were obtained from Hubei Xinyangfeng Fertilizer Co., Ltd., Jingmen, China.The commercial organic fertilizer includes 45% organic matter content and more than 5% NPK content. NPK compound fertilizer is composed of N, PSoil samples of the surface layer (0–10 cm) were collected two years and four months after the final fertilization (in September 2021). Five sampling points in each plot were randomly selected, and soil samples taken from the five points were mixed together evenly. First, the plant roots, residues and break stones in the soil samples were removed. The samples were then passed through a 2 mm mesh sieve and divided into two sections: one was stored at −80 °C in a refrigerator for DNA extraction, and the other was used for the determination of soil chemical properties.4+–N) was assayed using a spectrophotometric method, with Nassi reagent acting as the extraction agent. Nitrate (NO3—N) in the soil was extracted with 2M KCl and measured by ultraviolet spectrophotometry. Available phosphorus (AP) content was extracted from soil samples with a mixed solution of ammonium fluoride and hydrochloric acid and determined using the visible spectrophotometer [The pH value of each soil sample was determined with an acidimeter  in a solution that was mixed with double-distilled water and soil (2.5:1). The content of soil organic matter (SOM) was measured using the potassium dichromate oxidation method . Ammoniuotometer ,43.−1 with sterile water to ensure amplification efficiency and accuracy. The V3–V4 hypervariable regions of bacterial 16S rRNA genes were amplified by polymerase chain reaction (PCR) with the forward primer (341F 5′-CCTAYGGGRBGCASCAG-3′) and the reverse primer (806R 5′-GGACTACNNGGGTATCTAAT-3′). The PCR amplification mixture contained 15 μL Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 0.2 μM forward primer, 0.2 μM reverse primer and 10 ng template DNA. The thermal cycling program of the PCR reaction process included an initial denaturation step at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, and then a final 5-min extension at 72 °C. PCR reactions were subjected to three replicates [DNA extractions from 0.5 g fresh soil samples were performed using the Magnetic soil DNA kit , referring to the manufacturer’s instructions. The extracted DNA concentration and purity were monitored on 1% agarose gels, and the concentration was diluted to 1 ng·μLplicates ,44.® Ultra™ IIDNA Library Prep Kit (Cat No. E7645) was used to sequence the construction of libraries following the manufacturer’s protocols, and the library quality was evaluated on the Qubit® 2.0 Fluorometer . Finally, the library was paired-end sequenced on an Illumina Novaseq platform  according to the standard protocols of Novogene Co., Ltd. . The raw data have been stored in the Sequence Read Archive (SRA) data of the National Center for Biotechnology Information (NCBI) database with accession number PRJNA820355.PCR products were mixed with an equal volume of 1 X loading buffer and detected by electrophoresis on 2% agarose gel, which was then purified using a Qiagen Gel Extraction Kit . NEBNexthttp://ccb.jhu.edu/software/FLASH/ 27 March 2022) [https://www.arb-silva.de/ 27 March 2022) and alpha diversity indexes were calculated in QIIME2 software (Version QIIME2-202006).For the raw reads, quality filtering was done using the fastp (Version 0.20.0) software and merged using FLASH (Version 1.2.11 ch 2022) . Then, tch 2022) . Amplicoch 2022) . ASV tax10 (X + 1) conversion. Significant differences in the relative abundance of dominant bacteria at phylum and genus levels were analyzed by the Kruskal−Wallis test. Principal Coordinate Analysis (PCoA) of weighted unifrac distances was used to explore the beta-diversity changes between bacterial communities for each sample. Analysis of Similarities  by Anosim function within QIIME2 software was used to test community structure discrepancies among different treatments. Linear discriminant analysis (LDA) and effect size (LEfSe) analysis were conducted to identify potential bacterial biomarkers of each treatment. Mantel tests  were used to test the relationship between the soil environment and the distribution of bacterial communities (at the ASV level). Redundancy analysis  and Spearman’s correlation analysis were applied to evaluate the influence of edaphic factors on the main bacterial phyla and genera.One-way analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test in SPSS25.0 software  was used to determine the differences in soil chemical properties and alpha-diversity indexes that were normalized by using a loghttp://www.rstudio.com/ 27 March 2022) and the correlation coefficient and significance level  was set up. Analytical data were the ASVs with the top 100 absolute abundance in each sample [http//:gephi.org/ 27 March 2022).Co-occurrence networks were used to explore the interactions between soil bacterial taxa (based on ASV levels). The network construction was based on Pearson’s correlation analysis by using the psych package in Rstudio software . The soil pH value in OCPF treatment was significantly higher than CK by 8.16%, and the SOM content also increased by 20.39% and 17.53% compared to CK and CPF, respectively. The average content of NH4+–N and NO3−–N in OCF, OCPF and CPF treatments were noticeably greater than those in CK. The content of NH4+–N and NO3−–N in OCF treatment were 152.94% and 90.59% greater than CK, respectively. The soil AP content showed an increasing trend with increased fertilization in the order: OCF > CPF > OPF > OCPF > CK.The combined fertilization enhanced the soil pH value and SOM content  showed that the sequencing depth effectively reflected the actual richness of soil bacterial communities . The aveR = 0.372, p = 0.005). The PCoA analysis explained 60.94% of the total variation in bacterial communities, of which PC1 accounted for 42.32%. However, soil bacterial communities were not clearly clustered into different groups according to the different fertilization treatments in the ordination diagram . Verrucomicrobia and Bacteroidetes (except for OCF) showed higher relative abundances in fertilization treatments than those in the CK group. The relative abundance of Firmicutes and Actinobacteria was increased in OCP, OCPF and CPF treatments, whereas Acidobacteria and Proteobacteria in OCPF and CPF treatments were less abundant than that of CK . Redundancy analysis between the relative abundance of bacteria and soil factors demonstrated that the soil chemical properties explained 21.36% and 40.23% of the total variation on phylum and genus levels, respectively (The Mantel tests suggested that soil pH and SOM have a strong effect on the structure of the bacterial community (ectively A,B. The ectively .4+–N and NO3−–N contents. The abundance of Nitrospirae was positively correlated with soil pH value, SOM, NH4+–N and NO3−–N contents. Most phyla such as Verrucomicrobia, Gemmatimonadetes and Nitrospirae were positively linked with soil pH value. The abundance of Gemmatimonadetes and soil AP content, Patescibacteria and SOM content were significantly positive correlations (Spearman’s correlation analysis of major bacterial phyla and soil properties showed that the relative abundance of Chloroflexi was significantly negatively related to soil pH value, NHelations A. For thelations B, Subgrop < 0.05, It is widely known teak grows better in rich soils with a neutral and slightly alkaline pH ,51. TeakThe increase in the soil pH value might also be related to the decomposition of soil organic matter . A signiSoil pH may play a major role in transforming soil nutrient contents by changing bacterial taxa. Microbial communities exhibit poor growth in lower pH environments, and a relatively acidic condition usually inhibits both enzyme activity and overall cellular metabolism ,60. A prFertilization had a positive impact on soil pH and nutrient levels, directly or indirectly influencing changes in bacterial community composition and abundance. In the present study, soil pH and organic matter were the main impact factors after fertilization in the teak plantation , which aThe application of organic fertilizer plays a substantial role in sustaining soil organic matter levels and simultaneously, the input of organic materials is also beneficial for the improvement of soil aggregate structure and available nutrients . These cBacteria are the most abundant and diverse group of soil microorganisms, maintaining the stability and sustainability of the soil ecosystem in forests ,71. The p < 0.01, Determining the variations in soil bacterial composition between fertilization versus no fertilization treatment can effectively assess the effect of soil quality, health and fertilization on artificial forest ecosystems . In the Co-occurrence network analysis provides evidence of the direct and indirect cooperation or composition relationships among microbial taxa . The intOur study found that fertilization altered the relationship between bacterial species, and soil bacterial communities in different treatments exhibited different patterns of co-occurrence networks . The numIn this study, mixed fertilization with organic, NPK and phosphorus fertilizers significantly increased the content of soil organic matter and available nutrients and reduced soil acidity in young teak plantations. The changes in organic matter level and pH value in soil further altered the composition and network of the bacterial community. The relative abundance of Gemmatimonadetes and Myxococcota in OCPF treatment was clearly enriched, while it was decreased for Chloroflexi, Acidobacteria and Proteobacteria. The topological properties of the co-occurrence network showed that the nodes, edges, network density and average degree were the highest in OCPF, indicating that the bacterial community structure in this soil was more stable and complex. Moreover, soil pH and organic matter were closely associated with bacterial species and were regarded as the primary factors shaping bacterial community structure. Further research on the impact of keystone species within microorganisms is required to identify their contribution to nutrient cycling and to predict the coupling relationship between their function and forest productivity in plantation ecosystems.


Prunus persica L. Batsch); however, an excess is toxic. In plants, amino acids are involved in responses to abiotic and biotic stresses, such as water deficit, extreme temperatures, high salinity, and heavy metal stress. However, the role of leucine in the regulation of heavy metal stress is currently unclear. Therefore, we investigated the effects of exogenous leucine on the growth of peach seedlings under Cu stress. Exogenous leucine improved the leaf ultrastructure and ionic balance and increased the chlorophyll content, the net photosynthetic rate, and the maximum photochemical efficiency. Furthermore, it attenuated Cu-stress-induced oxidative damage via a decrease in reactive oxygen species (ROS) and the regulation of the antioxidant and osmotic systems. These effects, in turn, ameliorated the reductions in cell viability, cellular activity, and biomass under Cu stress. Moreover, exogenous leucine increased the activities of nitrate reductase (NR), glutamine synthetase (GS), and glutamic acid synthetase (GOGAT) and thus improved the nitrogen metabolism efficiency of plants. In conclusion, leucine significantly improved the photosynthetic performance and antioxidant capacity, reduced Cu accumulation, and promoted nitrogen metabolism, which in turn improved the resistance of peach seedlings to Cu stress.Heavy metal contamination has a severe impact on ecological health and plant growth and is becoming increasingly serious globally. Copper (Cu) is a heavy metal that is essential for the growth and development of plants, including peach ( Moreover, the accumulation of Cu in the food chain can lead to poisoning in humans due to excessive consumption  × VT/(V0 × W), where VT = the total volume of the extract; V0 = the assay volume; and W = plant fresh weight.Lipid peroxidation can be measured by determining the malondialdehyde (MDA) content. The MDA determination followed the method described by Zhao  with min−1 protein. The CAT activity was measured by monitoring the decrease in H2O2 at 240 nm for 1 min at 25 °C. The 3 mL reaction mixture contained 100 μL of enzyme extract and 2.9 mL of sodium phosphate buffer  containing 10 mM H2O2. The CAT activity was calculated as the amount of enzyme that caused a reduction in the absorbance at 240 nm of 0.01 per minute [−1 cm−1 at 290 nm) for AsA. One unit of APX was defined as the amount of enzyme that breaks down 1 μmol AsA min−1. The guaiacol peroxidase (GPX) activity was measured using a modification of the procedure by Zhao [2O2, and 100 μL of enzyme extract. The increase in absorbance due to the oxidation of guaiacol was measured at 470 nm (E = 26.6 mM−1 cm−1). The dehydroascorbate reductase (DHAR) activity was determined according to Zhao [−1 cm−1). The enzyme activity was expressed as unit g−1 min−1 FW.All enzymatic activities were determined at 25 °C and expressed as U ghttps://www.ncbi.nlm.nih.gov/, accessed on 14 November 2022). RNA of leaves was extracted using the RNA Extraction Kit  and reverse-transcribed to cDNA (Takara). The primers used in this study are shown in –ΔΔCT method [Genes of the antioxidant system were identified from the NCBI database  reduction method and expressed as the amount of TTC reduced by per gram of root .The root electrolyte leakage was determined according to a previously described method . Approxi3-N) content of the leaves was determined by salicylic acid colorimetry [On the sixth day after the treatment, the nitrate–nitrogen (NOorimetry . Under a4+-N) content was determined by Nessler’s spectro-photometric method [The ammonium–nitrogen (NHc method . The leaThe nitrate reductase (NR) activity in the leaves was determined by sulfanilamide colorimetry . The leaWe determined the glutamine synthetase (GS) activity using the plant GS enzyme activity assay kit and determined the glutamic acid synthetase (GOGAT) activity using the plant GOGAT enzyme activity assay kit , following the manufacturer’s instructions.p < 0.05.We collected three biological replicates for each treatment. Origin version 9.8 was used to conduct all statistical analyses. Duncan multiple range tests, which are included in SPSS version 20.0, were performed to detect any statistically significant differences in the mean values . The threshold of statistical significance used for all tests was It can be seen from The ultrastructural changes in the cells of peach leaves under Cu stress and with exogenous leucine are shown in 2, so that the final value > 0 was positive regulation and otherwise it was negative regulation. We found that the relative gene expression of antioxidant protective enzymes in the control and Leu treatments was negative, indicating that the control and Leu treatments did not activate the antioxidant protective enzyme system. However, the Cu treatment and the Cu+Leu treatment could activate the antioxidant protective enzyme system in order to remove ROS in the plant. The relative expression of antioxidant protective enzymes in the Cu treatment with Leu addition was higher than that in the Cu treatment, indicating that Leu could reduce the production of ROS in the plant under Cu stress  could increase the chlorophyll content of ‘Hanfu’ apple seedlings and keep the leaves in a healthy state. However, when the Cu2+ concentration exceeded 300 mg·kg−1, the plants were stressed, and the chlorophyll content and photosynthetic efficiency of seedlings decreased [−1 Cu2+, indicating that a high Cu concentration seriously inhibited the synthesis process of chlorophyll and thus reduced plant photosynthetic efficiency [Plants are inevitably challenged by various environmental stresses, in particular salt, heat, intense irradiance, and heavy metal stress. Abiotic stress can reduce crop growth, plant leaf area, and photosynthesis rates . Leaves,ecreased . Under Cecreased . Chloropficiency . The maxficiency . In thisPotamogeton malaianus under Cu stress [2+ can promote plant photosynthesis, but an excessive concentration of Cu2+ will inactivate chlorophyll proteins, change the chloroplast ultrastructure, destroy the structure and function of thylakoids, ultimately inhibit photosynthesis, and seriously affect nutrient accumulation [The morphology and ultrastructure of chloroplasts also directly affect the photosynthetic performance of plants. Several studies have investigated the effect of Cu on the chlorophyll ultrastructure ,36. Lin u stress . An apprmulation . In this2-), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH-), which negatively elicit oxidative stress on cellular structures and metabolism [PpCAT, PpSOD, and PpGPx were more upregulated. Under Cu stress, the expression of antioxidant genes and the activity of antioxidant enzymes were increased after adding Leu, indicating that Leu could reduce the peroxidation damage under Cu stress  and the regulation of the antioxidant and osmotic systems. These effects in turn ameliorated cell viability and biomass accumulation with improved resistance of peach seedlings to Cu stress. This study illustrates that leucine alleviates the damage caused by Cu stress in peach seedlings, which may provide more reference data for environmental risk assessments of Cu and make it possible to reuse soils with excessive copper contents caused by heavy metals.


Introduction: Short tandem repeats (STRs) have been used for various identity typing methods worldwide. They have high discrimination power in human identification in forensics, paternity testing, missed personal identification, genetic diseases, and gene regulatory functions. They have also been used to detect and monitor the stability of diseases, including various types of cancer. This study aimed to investigate the impact of leukemia on the detection and stability of STR markers.Methods: DNA was isolated from 30 participants (15 with chronic myeloid leukemia( CML) and 15 healthy controls) and used to amplify STR markers using specific primers.Results: We found that the blood of those with leukemia had more 9.3 and 9 alleles at the tyrosine hydroxylase 1 (TH01) marker than the blood of the healthy control samples. The results of this study will help researchers understand leukemia’s effect on the detection and stability of STR markers in leukemic patients compared to healthy individuals.Conclusion: Our results demonstrate that STR markers could become useful in genetic studies of leukemia cases. Microsatellites also known as short tandem repeats (STRs), are small DNA sequences (two to six base pairs) with multiple repeats that make up around 3% of the human genome . The numThe STRs have become efficient tools for estimating polymorphism over biochemical and other markers because of several distinct features, including the low required DNA amount and a high number of alleles -9. The SAfter the publication of the first draft of the human genome , numerouDetection and estimation of STR markers' frequency is a preliminary step for various populations/subpopulations or different countries prior to their use in various applications. Different countries have conducted such studies including the USA ,41, TurkThe STR markers are used to monitor chimerism status in hematopoietic stem cell transplantation (HSCT), an essential process for transplant rejection. Also, it monitors the chimerism percentage and stability of STR markers in leukemic patients after transplantation -52.The instability of STR markers in cancerous tissues could occur because of defects in the DNA repair pathways and the accumulation of alterations in microsatellite loci. The STR markers exhibit instability in various cancers, including lung , papillaIn Saudi Arabia, few studies have been conducted on STR markers to detect allele variations in subpopulations as genetic diversity of forensic STR markers -64. The Study design, ethics statement, and human participants The current research is a bioethical case-control study. We obtained the approval of the Dean of Scientific Research at Taif University in the Kingdom of Saudi Arabia as well as that of the King Salman Medical City Institutional Review Board . Participants signed consent forms before blood samples were collected at the King Fahad Hospital's Department of Oncology and Hematology in Medina, SAU. Furthermore, the control samples selection criteria are based on those who donate blood voluntarily, are healthy, and have normal complete blood counts while the leukemia case selection criteria are based on diagnostic tests.Case selection, sample collection, and sample sizeA total of 30 participants (15 with chronic myeloid leukemia( CML) and 15 healthy controls) were included in the study. All participants were aged 35 to 61. Of a total of 15 CML cases, only three  had undergone chemotherapy treatment. The analyses were performed on blood samples placed on Flinders Technology Associates (FTA) cards. DNA extractionThe DNA was extracted from blood with a PrepFiler Express™ Forensic DNA Extraction Kit, which was used according to the manufacturer’s instructions . The extracted DNA samples were fluorometrically quantified with a Quantifiler™ Human DNA Quantification Kit , which was used according to the manufacturer’s instructions.DNA amplification Amplification of the 16 STR loci , D5S818, and FGA was performed with an AmpFLSTR™ Identifiler™ Kit Plus PCR Reagents , which was used according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was carried out with a Gene-Amp® PCR 9700 thermal cycler ; initial incubation was at 95 °C for 11 min, followed by 30 cycles of denaturation , annealing , and extension .STR genotypingCapillary electrophoresis was performed using POP-4™ Polymer (Applied Biosystems) in an ABI Prism 3500 Genetic Analyzer (Thermo Fisher) under default conditions  with the following components per sample: 12 μL Hi-Di™ Formamide (Thermo Fisher), 0.5 μL 500 LIZ™ Size Standard (Applied Biosystems) and 0.5μL PCR product. Prior to fragment analysis, this mixture was heat-denatured for seven minutes at 95 °C and snap-cooled on ice for another three minutes, after which it was transferred onto a plate and placed in the genetic analyzer.Genetic evaluationSpecimens were evaluated using the specialized software, GeneMapper® IDX version 1.4 (Thermo Fisher Scientific). The analytical and stochastic thresholds were set to 50 RFU and 200 RFU, respectively. The profiles obtained from the blood samples of the leukemic patients were then compared to those from the healthy individuals to observe the difference in genetic information.Statistical analysisData analysis was performed using Statistical Package for Social Sciences (SPSS) version 26 . A two-way contingency table was created to investigate whether associations existed between different alleles and leukemia. Two alleles  were used as predictors of leukemia. Because of the small sample size, the use of contingency tables larger than 2 × 2, and the presence of 2 cells with an expected count < 5, the Fisher-Freeman-Halton exact test was performed. The odds ratio (OR) and confidence interval (CI) were also calculated.Thirty samples collected from 15 patients with leukemia and 15 healthy individuals were used in this study to investigate the stability and detection of 15 somatic STR markers and the amelogenin (AML) marker. All markers were successfully detected in all participants. Three markers-D21S11, TH01, and D19S433-showed microvariants . Table Interestingly, the TH01-9 and the micro variant 9.3 were detected more in the leukemia cases. Therefore we focused on comparing allele 9 and the microvariant allele 9.3 between normal and leukemic cases as shown in Figures In recent years, reports of the links between genetic markers in populations and diseases have increased. Several studies have used forensic STRs as genetic markers to look for cancer-related alleles. Furthermore, many studies have reported a strong association between TH01 polymorphism and its related alleles in several diseases compared to control groups. The present study, even with its small sample size, is in concordance with research ; Qi et aAccording to the GenBank top strand nomenclature, TH01 is situated inside the first intron of the human tyrosine hydroxylase gene, and the chromosomal location is on 11p15.5. Frequently defined by the repeat patterns [AATG]n or [TCAT]n as allele 9 [AATG]9 while allele 9.3 [AATG]6ATG[AATG]3 ,69. The Researchers have found an important link between changes in TH expression and the development of neurological, psychiatric, and cardiovascular diseases ,71. Wei Our DNA profiling showed a strong link between TH01  and the presence of leukemia. Despite the limited sample size, this finding confirms the association between the allelic configuration of STR markers and CML . AdditioThis study presented the genotype of leukemia cases as a piece of significant evidence. The predominance of allele‐9 at the TH01 locus (AATG repeat unit) in leukemia cases indicates an association with leukemia. Furthermore, the TH01 microsatellite combined with alleles 9 is a good biomarker candidate for leukemia. While microvariant allele 9.3 was found in leukemia cases, it was not significant due to the small sample size. However, STR markers could become useful in genetic studies of leukemia cases. Finally, forensic STR markers may provide more information than just identity.


We proposea systematic, high-throughput computational approach primarily aimedat identifying API/coformer pairs that are unlikely to lead to experimentallyobservable cocrystals and can therefore be eliminated with only abrief experimental check, from any experimental investigation. Onthe basis of a well-established crystal structure prediction (CSP)methodology, the proposed approach derives its efficiency by not requiringany expensive quantum mechanical calculations beyond those alreadyperformed for the CSP investigation of the neat API itself. The approachand assumptions are tested through a computational investigation on30 potential 1:1 multicomponent systems  involving3 active pharmaceutical ingredients and 9 coformers and one solvent.This is complemented with a detailed experimental investigation ofall 30 pairs, which led to the discovery of five new cocrystals  and a cis-aconitic acid polymorph. The computational approach indicates that,for some APIs, a significant proportion of all potential API/coformerpairs could be investigated with only a brief experimental check,thereby saving considerable experimental effort.Controlling the physicalproperties of solid forms for active pharmaceuticalingredients (APIs) through cocrystallization is an important partof drug product development. However, it is difficult to know  Experimental and computational coformer screens are performedin parallel, for the combination of 3 active pharmaceutical ingredientsand 10 coformers. The experimental screen generates five new cocrystalforms and one new pure form, while the computational methodology,which allows for reuse of quantum mechanical calculations, is demonstratedto have a dramatic increase in efficiency for studying cocrystals,if the pure forms have already been investigated. The issuewas resolved after four years of efforts by reformulating the APIas a cocrystal with lopinavir. Cocrystals have increasingly been usedin pharmaceutical products, including Depakote , Cafcit (caffeine and citric acid), Lexapro , Odomzo (sonidegib and diphosphate), Suglat (prolineand ipragliflozin), Entresto ,and Steglatro (ertugliflozin and l-pyroglutamic acid).3Inthe notorious case of Ritonavir,8 but screening of multiple API/coformer pairs can be time-consuming,expensive, and, in the early stages of a pharmaceutical developmentworkflow, limited by a lack of sufficient amounts of API material.Thus, computational methods for the rapid and reliable assessmentof the likelihood that a given API/coformer pair may form a stablecocrystal could significantly accelerate formulation development andmitigate the risks associated with the neat API solid form being unstableor insoluble. Given an API and a set of potential coformers  list9 or the Everything Added to Food list10), a computational screen would aim to determine which ofthese coformers are likely to lead to thermodynamically stable cocrystalsfor a given API. Those could then be studied experimentally to verifythe existence or not of the predicted cocrystals.Despite the potential contribution of cocrystalstoward the developmentof improved drug products, the identification of suitable coformersfor a given API remains a complex process. Experimental screeningmethods have been effective in several investigations,13 or of molecular electrostatic potential interactions14 through molecular complementarity methods.15 Further, the electron density of molecular components,combined with statistical thermodynamics in the COSMO-RS package,16 has been used to predict the cocrystallizationpropensity of selected APIs with libraries of coformers.17 Approaches based on machine learning methodsusing molecular descriptors18 have alsobeen proposed. However, none of these methods consider explicitlythe crystal environment and its effects on the stability of a proposedcocrystal. This deficiency can be addressed to some extent by crystalstructure prediction (CSP) methods, the reliability of which has increasedsignificantly20 over the past 30 years.Computationalcoformer screens presented in the literature haveemployed a number of different approaches. Some are based on the interactionsof API/coformer dimers in the gas phase through calculations eitherof hydrogen bond propensity between API and coformer24 Cocrystals are particularly challenging in this context becauseintroducing more molecules into the crystal’s asymmetric unitincreases the number of degrees of freedom that need to be exploredin identifying all low-energy crystal structures. Thus, the de novo generation of a computational landscape for a givenpair of compounds with state-of-the-art methods can often take hundredsof thousands or even millions of CPU hours.26CSP methods have already been used successfully to study cocrystalsin several investigations.27 or XtalPi,28 the force field is tailoredto the system under consideration by carrying out periodic dispersion-correcteddensity functional theory (DFT-d) calculations29 on a set of crystal structures. Thus, for each API + coformerpair, an initial set of cocrystal structures must be generated, followedby an expensive DFT-d calculation for each such structure. By contrast,in the CrystalPredictor30/CrystalOptimizer31 framework, isolated-molecule in vacuo quantum mechanical (QM) calculations are carried out separatelyfor each component of the crystal. The results of such QM calculationsat different molecular conformations are used to construct local approximatemodels (LAMs)31 that can compute the intramolecularenergy (ΔUintra) and electronicdensity (in terms of atomic charges and multipoles) as explicit, computationallycheap functions of molecular conformation. For moderate changes inmolecular conformation around the reference point at which the correspondingQM calculations are performed, it has been shown that it is possibleto derive a set of LAMs that collectively can provide QM-like accuracyin describing intramolecular energy, electrostatic potential, andgeometry.32Another challenge for the application of CSP methods to cocrystalsrelates to the derivation of sufficiently accurate lattice energymodels (force fields) for a relatively large number of API + coformerpairs. In some CSP methods, such as GRACE35 and at therefinement stage.31 In theformer case, they are constructed a priori at selectedsets of points organized in uniform or nonuniform grids over the molecularconformational space. At the refinement stage, they are constructed“on the fly” as new areas of conformational space areexplored by the lattice energy minimization algorithm. At both stages,LAMs are stored in a database from where they can be retrieved inorder to avoid repeating QM calculations at the same or similar molecularconformations.LAMs are used at boththe global search stage of a CSP investigationsame LAMdatabase can be used in assessing the likelihood of cocrystal formationagainst multiple coformer candidates. Moreover, since the set of acceptablecoformers for applications such as pharmaceuticals is fixed, the LAMdatabase for each one of these coformers can be constructed once andfor all, for subsequent use with any API of interest.Because of the computational cost of the QM calculations,constructingthe LAM database is typically the most expensive element of a CSPinvestigation. However, in the context of applying CSP to cocrystalscreening, the above approach can lead to dramatic cost reductionsby taking advantage of the fact that a given compound’s LAMdatabase is independent of crystal structure or of any other compoundin the lattice. Thus, for a given API, the Buildingon the above ideas, in this paper, we propose a systematicand efficient approach for performing a computational coformer screenfor a given API and a set of candidate coformers, based on multipleCSP investigations carried out in parallel. We test the reliabilityof this approach by also carrying out experimental coformer screensusing a variety of approaches chosen to achieve a thorough search and one that could be carriedout in an industrial context.The approaches used for studyingcocrystals computationally andexperimentally are outlined in 2For the purpose of this article, we will consider standardizedcomputational and experimental methods that are accurate yet affordable,allowing them to be adopted within the context of current pharmaceuticalpractices. These are outlined below.2.135 The flexible conformational degreesof freedom are determined based on the changes in intramolecular energyvalues arising from ±15° perturbations applied to thosetorsional angles that were identified as potentially flexible by thevalues of second derivatives at the gas-phase conformational minimum.Isolated-molecule QM calculations are performed in Gaussian 09 atthe PBEPBE level of theory using the 6-31G basis set. Startingwith an initial uniform LAM grid, the adaptive LAM algorithm32 is run until convergence is achieved, with theconvergence criterion Δ* equal to 5 kJ/mol. The set of parametersreferred to as the “FIT potential” is used to describethe exchange-repulsion and dispersion interactions.39 In the global search space, 500 000structure minimizations are performed, sampling the 59 most commonspace groups.CSP calculationsfor all systems are carried out using the code CrystalPredictor version2.4.3.40After the CrystalPredictor calculations are completed,a final clustering of generated structures is carried out with theCOMPACK algorithm. All unique structures within 20 kJ/mol (to a maximumof 500 structures) of the global lattice energy minimum from thisprocess are refined using CrystalOptimizer 2.4.5. The latter makesuse of the results of QM calculations performed at the PBE1PBE/6-31G)level of theory; additional flexibility is introduced , and electrostatic interactionsare described by atomic multipoles. Repulsive/dispersive interactionsare described by the DB2021 set of parameters, which were fitted toreproduce periodic DFT geometries and energies across an extensiveset of crystal structures of organic molecules, at the same levelof theory.2.22.2.1t-butyl-4-hydroxyanisole(Aldrich), nicotinic acid (Bayer), nicotinamide (Merck), oxalic acid(Merck), succinic acid (Fluka), and cis-aconiticacid (Aldrich). Pyridine, chloroform, and n-heptanewere of p.a. grade (>99.8% pure) and purchased from Merck or VWR.Work was done on commerciallyavailable samples (purity > 98%) without further purification:carbamazepine, acetylsalicylic acid , acetaminophen, pyridoxine (Sigma), methyl paraben (Merck),propyl 4-hydroxybenzoate (Merck), 3-2.2.2Where the thermal stability and relative differencesin melting points allowed, the contact preparation method was usedto investigate cocrystal formation. An Olympus BH2 polarization microscope equipped with a Kofler hot-stage was used. Cocrystals were preparedon the hot-stage by first melting the higher-melting compound on amicroscopic slide covered by a glass slide and subsequently coolingit down to create a thin crystal film. The lower-melting compoundwas then melted on the same microscopic slide. Through capillary action,the liquid was drawn below the cover slide until it reached the higher-meltingcoformer and then rapidly cooled. This gave rise to the possibilityof a crystalline film of the cocrystal forming at the zone of mixing.2.2.3Mixtures ofa 1:1 molar ratio were prepared and transferred to small vials. Anamount of 300–600 μL of solvent was added to 200 mg ofthis mixture, and the slurry was stirred in the temperature rangefrom 10 to 30 °C for up to 4 weeks. Samples were drawn periodically,for the first week on a daily basis, and then every week, and analyzedwith PXRD.2.2.4Mixtures of a 1:1 molar ratio were prepared and thoroughlygroundusing an agate mortar and pestle. One to two drops of solvent wereadded to an amount of 100 mg, and the paste was subsequently milledin five stainless steel vessels with two balls of the same materialand 0.5 cm in diameter using a Retsch ball mill MM 301 at 30 Hz for 15 min. The resulting powders were analyzed with IR spectroscopyand PXRD.2.2.51:1 molar mixturesof the APIs and coformers were dissolved at room temperature and transferredinto vials with pierced lids, and then the solvent was allowed toslowly evaporate.2.2.6Equimolaramounts were prepared, thoroughly ground using an agate mortar andpestle, and transferred into 0.3 mL vials. The sublimation experimentswere carried out under elevated temperatures at a pressure of 200mbar with the aid of a CrystalBreeder .2.2.7α1,2 radiation source with a focusingmirror, a 0.5° divergence slit, a 0.02° Soller slit collimatoron the incident beam side, a 2 mm antiscattering slit, a 0.02°Soller slit collimator on the diffracted beam side, and a solid-statePIXcel detector. The patterns were recorded at a tube voltage of 40kV and tube current of 40 mA, applying a step size of 2θ = 0.013°with 40 or 80 s per step in the 2θ range between 2° and40°.PXRD patternswere obtained using an X’Pert PRO diffractometer  equipped with a θ/θ coupled goniometerin transmission geometry, programmable XYZ stage with a well plateholder, Cu–K2.2.841 as implemented in the DASH structure solution package.42 Pawley fits43 andRietveld refinements44 were performed withTopas Academic V5.45 Simulated annealingwas used to optimize PBE-TS models against the diffraction data setsin direct space. The internal coordinate (Z-matrix) descriptions ofthe molecules were derived from computed structures (see section 3.1of the Supporting Information). The structureswere solved using 100 simulated annealing runs of 2.5 × 108 moves per run as implemented in DASH, allowing external andinternal degrees of freedom. The best solutions returned were usedas a starting point for PBE-TS fixed unit cell structure optimizationsusing CASTEP.46 The optimized structureswere then used as the starting points for rigid body Rietveld refinementsin Topas. For more details, see section 3.5 of the Supporting Information.Selected diffraction patterns were indexed withDICVOL04 usingthe first 12–20 peaks, and the space group was determined basedon a statistical assessment of systematic absences,3c = 1, ..., N, we assess the likelihoodof a cocrystalof stoichiometry APIncm being observed experimentally by consideringthe free energy difference quantity ΔΔGc defined asGncm],min,min and Gc],minfus[ at thistemperature:Gc]fus,min for either the lower-energystructure determined by the CSP study at step 1 or the most stable structure that has been observed experimentally.Evaluate lattice energy (3)c = 1 to N:For each coformer (a)c by performing a CSPstudy on that molecule.If not already available,developa LAM database for coformer (b)Uc],min,min,min and Uc],min,min and Uc],min,min and Uc],min[ in U values. However, the aspirin/carbamazepine cocrystal (TAZRAO) hasa ΔΔU above +5 kJ/mol. Overall, in decidingthe scope of an experimental screening program, a higher cutoff mightbe appropriate in recognition of the increased uncertainty that arisesfrom relying solely on computational data.The results presented in 4.5The computationalcosts of the neat API and cocrystal investigations are summarizedin Note that all of the computationalsteps are highly parallelizable:the QM calculations for generating LAMs at different molecular conformationsprior to the global search may be performed in parallel; the globalsearch involves the generation of hundreds of thousands of candidatecrystal structures and their subsequent lattice energy minimization,and these can also be carried out in parallel; the final refinementof hundreds of low-energy crystal structures can also be parallelized.The CrystalPredictor and CrystalOptimizer codes used for the CSP studiesfully exploit this potential for parallelizability. Additionally,for any given API, the CSP studies for cocrystals using differentcoformers can also be performed in parallel if sufficient computationalresources are available.In our calculations, we employed computationalclusters comprising32 cores for the LAM generation and refinement calculations, and 512cores for the global search. This allowed each CSP study for a neatAPI to be completed within about 3–5 days of wall time. TheCSP study for each API/coformer cocrystal took 2–3 days ofwall time. Moreover, given sufficient computational resources, multiplepotential cocrystals for the same API could be investigated in parallel.Overall, these statistics indicate that computational screening couldprovide useful input to drug product development on an acceptabletime scale.5U between the lattice energy of the most stable cocrystaland the sum of the lattice energies of the most stable neat formsof its constituents. An ab initio crystal structureprediction (CSP) methodology is used to determine the most stablecocrystal form. For the API and coformers, the most stable form mayeither be determined in the same manner or already be known experimentally.This paper has presented a computational procedure for cocrystalscreening for a given active pharmaceutical ingredient (API) againsta set of potential coformers. The assessment of the likelihood offormation of a stable cocrystal is based on the difference ΔΔA key characteristic of the proposed approach is its computationalefficiency. This is achieved by eliminating the need for any isolated-moleculequantum mechanical calculations beyond those already required forthe neat API and neat coformer CSP studies. Overall, this allows theefficient screening of relatively large numbers of coformers.U exceeding 5kJ/mol are unlikely to form cocrystals that can be obtained experimentally.This provides a useful criterion based on which unnecessary experimentalinvestigations can be avoided, thereby resulting in significant savingsin both time and API material. Future plans will focus on using thetechnique on larger and more flexible APIs that are of more relevanceto modern pharmaceutical developmental workflows.An investigation involving the application of the proposed computationalmethodology to three different APIs, each screened against 10 potentialcoformers was carried out. This was complemented by a parallel experimentalinvestigation on the same systems, aiming to identify as many cocrystalsas possible using a variety of experimental techniques. A comparisonof the results of the two investigations indicates that API/coformerpairs with a value of ΔΔ