Seafood products are one of the most perishable foods, and their shelf life is limited by enzymatic and microbial spoilage. Developing methods to extend the shelf life of fresh fish could reduce food waste in the fishery industry, retail stores, and private households. In recent decades, the application of lactic acid bacteria (LAB) as bioprotective cultures has become a promising tool. In this study, we evaluated the use of four starter cultures, previously selected for their properties as bioprotective agents, for sea bass and sea bream burgers biopreservation. Starter cultures impacted the microbial populations, biochemical parameters , and sensory properties of fish burgers, during 10 days of storage at 4 °C and then 20 days at 8 °C in modified atmosphere packaging (MAP). Also, storage time influenced the microbial and physicochemical characteristics of all the tested samples, except for TVB-N values, which were significantly higher in the uninoculated burgers. The volatilome changed in the different treatments, and in particular, the samples supplemented with starter presented a profile that described their rapid growth and colonization, with the production of typical molecules derived from their metabolism. The addition of bioprotective cultures avoided bloating spoilage and improved the sensory parameters of the burgers. The shelf life of the fish burgers supplemented with starter cultures could be extended up to 12 days. To increase fish consumption, two strategies are necessary: continuous training in school and the transformation of processed fish into derived products. The first strategy is very important because the preferences formed in childhood normally continue into adulthood, and for this reason, at each level of school, meal programs must include fish because they can contribute to the formation of healthy food habits [Fish are considered a functional food because of their high nutritional value, characterized by presenting components, such as mineral salts, high biological value proteins, and fatty acids, principally the polyunsaturated fatty acids of the omega 6 and omega 3 groups ,2. All td habits ,4.The second strategy is more important than the first. It is well known that people sometimes give up eating fish due to the presence of fish bones and the characteristic fish odor. Therefore, fish derivatives such as burgers can be a real strategy to increase fish consumption. Indeed, burger production allows the elimination of fish bones and a decrease in the characteristic odor of fish, which are the main barriers to fish consumption , while mFish are a very highly perishable product with a shelf life  limited by microbial growth ,10. ConsThe short shelf life of fresh seafood is often because of the growth of specific spoilage organisms [SSOs) ,13,14. T2, peroxide, and bacteriocins [The use of natural microbiota and/or their antimicrobial products as a biopreservation method is a recent and interesting approach to improve microbial food quality and safety ,27. Seleeriocins .The antagonistic effect against spoilage or pathogenic microorganisms is obtained either by directly adding living cultures or purified antagonistic substances or fermentation products ,12,28,29The aim of the present study was to develop fish burgers made with a mix of sea bass and sea bream meat and to improve their shelf life by bioprotective cultures. Carnobacterium divergens, C. maltoaromaticum and L. sakei; LAK-23—L. sakei; and FP-50—C. divergens, C. maltoaromaticum) were freeze-dried in a foil pouch. F-106 (Lacticaseibacillus casei) was derived from the Collection of the Department of Agricultural Food, Environmental, and Animal Sciences of the University of Udine and was grown in de Man, Rogosa and Sharpe (MRS) broth . After its growth, the strain was harvested by centrifugation at 8000× g for 10 min and then diluted in peptone water. The commercial starters were thawed, homogenized, and diluted in sterile peptone water  at the time of use. To evaluate the load of each starter, dilutions were performed in sterile peptone water, and 0.1 mL of each dilution was inoculated in the de Man, Rogosa and Sharpe (MRS) medium  by the double layer method for the LAB and in TSM agar  in a jar prepared for anaerobic reaction with a gas-packing anaerobic system  for the carnobacteria. The plates were incubated at 37 °C for 48–72 h, and the grown colonies were counted. Each suspension contained, on average, approximately 11 Log CFU/g.The commercial starters (BOX-57—Dicentrarchus labrax) and sea bream (Sparus aurata) weighing approximately 474–578 g and 404–440 g, respectively, were headed, gutted, and filleted. Fillets were then minced, mixed, and divided into five equal batches. Each burger was composed of 68% minced fish (50% sea bass and 50% sea bream), 20.5% potato, 5.5% water, 3% rice flour, 2% vegetable fiber, and 1% NaCl. The first batch was formed into patties and directly packaged and used as a control (CTRL). The other batches were inoculated with starter cultures at a final concentration of 105 CFU/g of burgers before being formed into patties. Four batches were prepared with each commercial and F-106 starter culture. The burgers were packed in MAP, consisting of 60% N2 and 40% CO2, and placed inside rectangular trays made of PET/PE/EVOH/PE: PET /EVOH /PE (Polyethylene), ANTIFOG—EVOH . The trays were laminated with a top film consisting of APET/PE/EVOH/PE: Amorphous Polyethylenterephtalat/PE (Polyethylene)/EVOH /PE (Polyethylene).Sea bass  and 2/3 at T2 (8 °C) (abuse temperatures).Burgers were analyzed at days 0, 6, 12, 18, 24, and 30 in triplicate to test microbial growth, physico-chemical characteristics, and sensory analyses.Carnobacterium spp. in TSM agar incubated at 30 °C for 2 days in anaerobiosis, (iv) Enterobacteriaceae in Violet Red Bile Glucose agar , incubated at 37 °C for 48 h; all data are expressed in log CFU/g product. L. monocytogenes was detected according to the ISO method [L. monocytogenes produce typical blue-green colonies surrounded by an opaque halo), and Salmonella spp., according to the ISO method [Salmonella). To confirm the growth of the starters, 5 colonies were collected from deMan Rogosa Sharpe plates and TSM agar and then identified by the methods reported in Iacumin et al. [Samples were analyzed by traditional microbiological methods to determine the main microbial population. Fifty grams of each sample was transferred into a sterile stomacher bag, and 100 mL of saline-peptone water  was added and mixed for 3 min in a stomacher machine . Further decimal dilutions were made in the same solution, and the following microbiological analyses were performed in duplicate agar plates: (i) total bacterial count (CBT) in Gelysate agar  incubated at 30 °C for 48–72 h, (ii) lactic acid bacteria (LAB) on double layer MRS agar , incubated at 30 °C up to 5 days, (iii) O method   was measured with an Aqua Lab 4 TE , and TVB-N  was measured according to Pearson [w/v). The distillate was collected in a solution of boric acid and titrated with sulfuric acid in the presence of methyl red. Data are expressed in mg Nitrogen/100 g. Th thiobarbituric acid value (TBARS) was determined directly by spectrophotometric quantification of compounds obtained by the distillation of a mix consisting of distilled water (50 mL) and fish product (10 g), acidified with hydrochloric acid  until pH 1.5. Then, 5 mL of the distillate was treated with 5 mL of a solution of thiobarbituric acid (TBA), obtained by mixing TBA (0.2883) in acetic acid (90%), and placed in boiled water for 35 min. After cooling, the solution was read at 538 nm. Three analyses were performed at each sampling point and data are expressed in nmol malonaldehyde/g.The pH was detected at 3 different points using a pH meter  by inserting the probe directly into the product. The water activity  using an Agilent Hewlett–Packard 6890 GC gas chromatograph and a 5970 MSD MS detector  equipped with a Varian (50 m × 0.32 mm × 1.2 μm) fused silica capillary column. Samples (3 g) were placed in 10 mL sterilized vials, added to a known amount of 4-methyl-2-pentanol  as an internal standard, and sealed by PTFE/silicon septa. The samples were heated for 10 min at 45 °C and then a fused silica SPME fiber covered with 85 μm Carboxen/Polydimethylsiloxane (CAR/PDMS)  was introduced into the headspace for 40 min. Adsorbed molecules were desorbed in the gas chromatograph for 10 min. The conditions were the same as those reported by Montanari et al. . VolatilThe sensory evaluation panel consisted of 12 nonprofessional assessors. The cooked burgers were presented on white plates at room temperature. Ten burgers of the control and of each treatment were evaluated. Assessors were asked to evaluate the following descriptors: odor , taste , flavor  and appearance (slime). The 12 assessors evaluated the presence or the absence of each of the nine descriptors. The results stated for each sample is the sum of the assessors who considered the presence of the descriptor out of the total of the assessors ,37. Thenp < 0.05.Statistical elaboration was carried out by the specific software Statistica for Windows, Version 8.0 . Means and standard deviations were calculated, and data were elaborated by principal component analysis (PCA) and by factorial ANOVA (two factors: starter culture and time) and Tukey’s HSD test. Significant differences among samples were calculated at To evaluate the capability of the different starter cultures to compete with the autochthonous microbial flora of the burgers, viable counts were performed during storage. The results of viable counts of total bacterial count (CBT), Enterobacteriaceae, and lactic acid bacteria (LAB) are reported in p > 0.05) of Enterobacteriaceae between the control and inoculated samples were recorded from Day 0 to Day 18 of storage. Therefore, burgers inoculated with LAK-23 presented low population amounts compared to the other samples. Listeria monocytogenes and Salmonella spp. were not detected in any of the samples. All pH profiles demonstrated clear acidification as a function of time, both in the control and when a starter culture was added. The initial pH of the burgers ranged from 6.17 to 6.31. On Day 12 of storage, the pH values decreased in all the samples, but the acidification in the inoculated samples was higher (p < 0.05) than that of the inoculated samples, which had pH values between 4.67 and 5.09. In contrast, at Day 18, the control, together with FP-50, had lower pH values. Subsequently, the pH values of all the samples remained practically unchanged until the end of storage (4.31–4.45). The water activity (aw) of the burgers at Day 0 was 0.9836. Total volatile basic nitrogen (TVB-N) is known as a product of bacterial spoilage and endogenous enzyme action, and its level is often used as an index to evaluate fish quality. The levels of these compounds, which increase with the onset of microbial spoilage, are primarily responsible for the fishy odors, which increase as spoilage proceeds (p < 0.05) between the samples were observed from Day 18. The increase in TVB-N values was lower in the samples inoculated with FP-50 starter culture. At the end of storage (30 days), the control samples had a higher value of TVB-N. The TVB-N content of all the samples exceeded the maximum level for acceptability for marine fish  at Day Principal component analysis (PCA) (data not shown) was performed to determine the relationship between design variables (time of storage and starter cultures) and microbial and physico-chemical variables. The samples on the same day of storage were grouped together, regardless of the starter cultures used. As expected, storage had a significant effect on the microbial population and physico-chemical properties of the burgers.The TBARS content changed slightly during storage. However, either at time 0 or at 30 days of storage, no significant differences were observed in any of the samples regardless of the treatment (with or without starters added). At 0 days, the TBARS means were at the level of 1.2 ± 0.3 nmol malondialdehyde/g in all the burgers, and then they slightly increased, reaching acceptable levels at 30 days .Alcohols, and in particular 1-propanol, isopropyl alcohol, and 1-penten-3-ol, were detected in higher amounts in the control at time 0. The second group was represented by aldehydes, among which hexanal, nonanal, and pentanal were the major contributors. All these aliphatic aldehydes derive mainly from lipid oxidation; they are responsible for “green notes”, but at higher concentrations, they can result in rancid perception .After 6 days, aldehydes demonstrated a small decrease in the control and in the fish burgers containing the culture F-106, while an increase was observed with BOX-57, FP-50, and LAK-23 samples. Alcohols were characterized by relevant increases, especially in the samples containing bioprotective cultures. These latter were also responsible for high concentrations of ketones, such as diacetyl and acetoin, especially in BOX-57, FP-50, and LAK-23. The same three bioprotective cultures were characterized by a higher accumulation of acetic acid, while the culture F-106 seems to confirm its slower metabolism under the conditions considered here.After 12 days, all the samples containing bioprotective cultures demonstrated higher amounts of aldehydes  than the control, while ketones were quite similar, except for F-106, which presented a relevant accumulation of diacetyl and methyl isobutyl ketone. Acetic acid was extremely low in the control compared with samples supplemented with bioprotective cultures, among which BOX-57 was the maximum producer and F-106 was characterized by a lower amount of this acid, confirming a slower metabolism of this strain in fish burgers. Alcohols increased, especially in the control and in the samples supplemented with F-106 and BOX-57. In addition to ethanol, the alcohol increase in the fish burgers containing bioprotective cultures was due to hexanol  and 1-octen-3-ol (which derives from the oxidative degradation of linoleic acid).The samples analyzed at 18 days were characterized by a drastic reduction in the proportion of aldehydes, mainly due to the decrease in hexanal. Additionally, ketones markedly decreased in the samples containing bioprotective cultures. In particular, in all these samples, the major decreases were observed for methyl isobutyl ketone, diacetyl, and acetoin. In contrast, this chemical group increased in the control, in which great proportions of diacetyl, acetoin, and 2-butanone were detected. The control was also characterized by a relevant increase in alcohol concentration, in particular attributable to the high proportion of ethanol and 3-methyl-1-butanol. Among acids, acetic acid was again the most important molecule, whose amount, despite an increase in the control, remained remarkably higher in the samples containing bioprotective cultures. Ethyl acetate was present in a higher proportion in the control. Considering the deep changes in the volatilome, which greatly influenced the sensorial aspects and the reaching of a high value of TVB-N, it was suggested to stop the VOC analysis at 18 days. To highlight the modification of the volatile profile during storage, Listeria monocytogenes, in different seafood products has been extensively studied. The growth of this pathogen was effectively limited by Latilactobacillus sakei in cold-smoked sea bass [Carnobacterium spp. in CSS [Leuconostoc gelidum and Lactococcus piscium in cooked and peeled shrimp and CSS [2 and the presence of CO2 limit or inhibit the growth of gram-negative bacteria such as Pseudomonas and Shewanella, considered the most important spoilage bacteria in the majority of seafood [Enterobacteriaceae can develop under these conditions and may play an important role in seafood spoilage [2 and are therefore often found as the dominant organisms in MAP fishery products [Enterobacteriaceae, yeasts, and endogenous LAB by the inoculation of C. divergens V41 in cold-smoked salmon (CSS) only when the initial natural microbial population was lower than 20 CFU/g. In fact, when it was more than 104–105 CFU/g, as in this study, no effect on the microbial population and physicochemical characteristics was observed. The similar counts of Enterobacteriaceae detected in the samples could also be related to the similar reduction in pH that occurred in all the samples, as previously mentioned. In fact, one of the main inhibitory mechanisms of Enterobacteriaceae by LAB is linked to a decrease in pH [Lactiplantibacillus plantarum 1.19 on the composition of Gram-positive bacteria of fresh tilapia fillets, but there was no effect on the Gram-negative bacteria. Heterofermentative LAB have been associated with bloating spoilage by the production of CO2 in several food products. Different species of the genus Leuconostoc were found to be responsible for bloating spoilage in vacuum-packed cooked meat and herring filets [Weissella have been related to bulging of broiler or cooked meat packages [2 observed in the control. The initial level of TVB-N in the fish burgers was relatively higher than that in similar products reported in other studies [Enterobacteriaceae counts, as reported in other studies [Seafood are one of the most perishable foods, and their shelf life is limited by enzymatic and microbial spoilage. In fact, the fish matrix is particularly suitable for the development of different types of microorganisms. In addition, during evisceration and filleting, microorganisms from the raw material, manufacturing environment, and human manipulation may contaminate the flesh . Severalsea bass , sea bresea bass , and colsea bass , Carnoba. in CSS , Leucono and CSS , just to seafood ,58. Howespoilage . Therefoproducts . Regardiproducts  observedse in pH . Anotherse in pH . Althougse in pH . Most lise in pH  observedg filets ,66. Membpackages . In thispackages . This ma studies ,68. More studies . The dif studies ,71. AccoThe medium level of TBARS was 2.1 ± 0.5 nmol malondialdehyde/g at 30 days, independent of the treatments; consequently, these values must be accepted. According to several authors , food prThe presence of bioprotective starter cultures and the length of storage changed the volatile profile of the fish burger when compared with the control. The most important accumulated molecules can be derived from the pyruvate metabolism of LAB. When fermentable sugars are scarce or absent, LAB shift to mixed acid fermentation, through which, starting from pyruvate, they can accumulate additional ATP following two different pathways: the pyruvate formate lyase and the pyruvate oxidase, with the production of acetate and ethanol as end products. In addition, the species used in these trials are facultatively heterofermentative and can ferment pentose sugars present; for example, in nucleotides, producing lactic, and acetic acid ,74. In aLacticaseibacillus casei (F-106) demonstrated slower kinetics, indicating a possible minor adaptation to the environmental conditions, particularly temperature (4–8 °C). The control presented a clearly different profile. Even if the most important molecules were the same, the kinetics of accumulation were delayed, and in contrast to other samples, after 18 days, ethanol was present in a higher proportion than acetate. These differences indicate that the LAB populations that colonized the burgers in the absence of bioprotective cultures were characterized by a different activation of metabolic routes during storage at refrigeration temperature.According to the results of PCA data, important differences in the volatilome were induced by the presence of bioprotective cultures. The samples supplemented with BOX-57, FP-50, and LAK-23 presented a profile that described the rapid growth and colonization of fish burgers by LAB, with the production of typical molecules derived from their metabolism. The strain Brochothrix thermosphacta without suppressing its metabolites. The panellists preferred the burgers inoculated with the starter cultures LAK-23 (L. sakei) and F-106 (L. casei). These results demonstrated that the inoculated starter cultures improved the sensory attributes of the fish burgers. Similar results were also reported by Comi et al. [The growth of the starter cultures could have had an inhibitory effect on the spoilage activity of the endogenous microorganisms. It was demonstrated that some LAB have the potential to suppress the metabolic activity of certain bacteria, limiting their spoilage potential . This tyi et al. , in whicThe results of this study indicated that the shelf life of sea bream and sea bass burgers, as determined by the sensory scores and physico-chemical and microbiological data, was 12 days. The inoculation of different starter cultures did not have a particular effect on the microbial populations and physico-chemical characteristics of the burgers.The volatilome changed in the different treatments, and in particular, important differences were induced by the presence of bioprotective cultures. The samples supplemented with BOX-57, FP-50, and LAK-23 presented a profile that described the rapid growth and colonization of fish burgers by LAB, with the production of typical molecules derived from their metabolism.The sensory attributes of burgers were affected by the presence of the bioprotective cultures, as the odors, flavors, and sticky, white slime indicative of deterioration were not observed. Additionally, inoculated burgers did not demonstrate bloating spoilage. The bioprotective cultures evaluated in this study can potentially extend the shelf life and improve the sensory properties of fish burgers, contributing to the reduction of food waste in the fish supply chain.


In recent years, sodium-glucose co-transporter 2 inhibitors (SGLT2i) have demonstrated beneficial renoprotective effects, which culminated in the recent approval of their use for patients with chronic kidney disease (CKD), following a similar path to one they had already crossed due to their cardioprotective effects, meaning that SGLT2i represent a cornerstone of heart failure therapy. In the present review, we aimed to discuss the pathophysiological mechanisms operating in CKD that are targeted with SGLT2i, either directly or indirectly. Furthermore, we presented clinical evidence of SGLT2i in CKD with respect to the presence of diabetes mellitus. Despite initial safety concerns with regard to euglycemic diabetic ketoacidosis and transient decline in glomerular filtration rate, the accumulating clinical data are reassuring. In summary, although SGLT2i provide clinicians with an exciting new treatment option for patients with CKD, further research is needed to determine which subgroups of patients with CKD will benefit the most, and which the least, from this therapeutical option. Chronic kidney disease (CKD) of any etiology is on the rise on a global scale. Caused by multiple conditions, such as diabetic kidney disease (DKD), hypertension, glomerulonephritis and various infections, in 2017, it was estimated to affect ~10% of the world’s adult population . The preSGLT2i received approval for treatment in T2DM nine years ago. In that period, they have evolved from a novel hypoglycemic agent, to a potent cardio- and renoprotective option in the T2DM and heart failure treatment arsenal, and recently, to an exciting standalone therapeutical prospect for CKD, regardless of the presence of T2DM. It all started with phlorizin, a natural product used to treat fever and infectious diseases, first isolated in 1835 by a French chemist ,7. AboutThis triggers the following question: what in fact makes the renal part of SGLT1/2 inhibition so attractive? Diving deeper into the mechanism, the glucose reabsorption in the kidney is facilitated by active sodium removal by the Na+/K+-ATPase on the basolateral membrane, which creates the electrochemical force for glucose entry via sodium driven sodium-glucose cotransport with the SGLT1/2 through the apical membrane ,11. GlucAfter almost a decade since the approval of the first inhibitor, the SGLT2i now represent a mainstay in the therapeutical guidelines of T2DM and HF. What is most interesting, and is in fact the focus of this article, is that SGLT2i have been widely shown to also provide substantial renoprotective effects, thus further expanding the plausible implications of this drug . Some auWith the mechanism of SGLT2i that occurs in the renal proximal tubule, the effects on the kidney itself, both direct and indirect, are unsurprisingly numerous .Firstly, SGLT2 inhibition reduces glomerular hyperfiltration, a pathophysiological mechanism that operates in T2DM, leading to the progression of CKD ,27,28. I2 homeostasis, verifying that diabetic kidneys displayed reduced baseline pO2 in both cortex and medulla, while SGLT inhibition by phlorizin improved cortical pO2, but reduced medullary pO2. The latter finding, interestingly, suggests a possible redistribution of active transport to distal nephron segments, resulting in the observed medullary hypoxia [Secondly, in order to fulfill its metabolic demand, the kidneys use up an unproportionally large amount of energy, as much as 10–15% of glucose . As earl hypoxia . Another hypoxia . An in vitro study by Ishibashi et al. further explained how SGLT2 inhibition ameliorates hypoxia, proposing that SGLT2-mediated glucose entry into the tubular cells is central to oxidative stress and inflammatory response that occurs in proximal tubular cells . An earlTo summarize the three broadly divided mechanisms, the first one, the reduction in hyperfiltration, and therefore glomerular injury, is DM-related through the TGF, but has also been speculated to be glucose-independent. The second, the reduction in energy consumption, and therefore hypoxia, is by definition glucose-dependent and greatly amplified in the presence of diabetes, although in theory, it occurs even in the absence of it. The third, the inhibition of inflammatory, fibrotic and proapoptotic response, to a significant extent does not necessarily relate to diabetes. This shows the great scope of SGLT2 inhibition’s direct effects on the kidney and provides explanations for its effectiveness in both diabetic and non-diabetic chronic kidney disease.Having discussed the direct effects of SGLT2 inhibition on the kidney at length, in this subsection, we will acknowledge the many indirect effects of SGLT2 inhibition on the kidney, whether this is through changes in hormones, metabolism or reduction in other risk factors for CKD. SGLT2 inhibition-induced glycosuria improves endogenous glucose production, pancreatic beta-cell function and insulin sensitivity. Consequently, it reduces insulin and increases glucagon secretion, shifting substrate utilization from carbohydrate to lipid ,60,61,62Multiple studies have reported other glucosuria-related effects of SGLT2i on multiple conditions that can have a profound impact on the kidneys, such as body weight and blood pressure. For example, obesity is known to increase renal blood flow and glomerular filtration, leading to albuminuria and long-term injury ,65. A grWith regard to blood pressure, multiple studies and meta-analyses have found a beneficial effect of SGLT2 inhibition on hypertension ,71,72,73circulus vitiosus, as CKD is known to decrease high-density lipoproteins (HDL) and increase triglycerides, while nephrotic syndrome is even more atherogenic by increasing low-density lipoproteins (LDL) and total cholesterol (TC) [Although dyslipidemia does not seem to have a direct impact on the progression of CKD, its detrimental effect on the kidney is funneled through damage to the cardiovascular system, which is, in fact, in many ways associated with CKD. Dyslipidemia and CKD, thus, form a sort of a rol (TC) . SGLT2 irol (TC) ,75,76.2 [SGLT2i have many protective effects on endothelial cells, such as promoting proliferation, migration and differentiation of said cells, while also increasing the bioavailability of nitric oxide derived from endothelium, which subsequently promotes vasodilation in multiple organs, including the kidneys itself, thus reducing the burden on the organ ,77. Hype2 . The putHematocrit and the reticulocyte levels increase with SGLT2i, an effect that was assumed to be a consequence of the diuretic effect. However, it is theorized that this occurs also because of the enhancement of erythropoiesis, which is a result of an increase in erythropoietin levels 80). Taken together, the increase in hematocrit during SGLT2 inhibitor therapy may indicate improvement of hypoxia and oxidative stress in the tubulointerstitial region of the renal cortex, as well as recovery of erythropoietin production by “neural crest-derived” fibroblasts . Taken t.As previously mentioned, SGLT2i have profound benefits on the cardiovascular system. SGLT2i have been observed to significantly lower the risk of not only renal, but heart failure as well ,87,88,89Over the last decade, there has been a great number of studies that have analyzed the clinical outcomes of SGLT2i on the kidney. A vast majority of them studied patients with DKD. Clinical evidence is summarized in 2. It found that SGLT2i attenuated the annual decline in eGFR and reduced the risk of composite renal outcomes [2 in patients started on SGLT2i, seemingly with no effect on the later outcomes [The EMPA-REG OUTCOME trial studied 6952 patients with DKD categorized into subgroups of albuminuria and eGFR, reaching a conclusion that empagliflozin significantly reduced kidney outcomes and reduced yearly loss of eGFR, which was consistent across all subgroups . A meta-outcomes . Anotheroutcomes . An exploutcomes . An addioutcomes ,97,98. Soutcomes . As prevoutcomes . A studyoutcomes . A lateroutcomes . Furtheroutcomes .Comparing dapagliflozin to a placebo over 6 weeks in patients with T2DM, a small randomized double-blind study found that dapagliflozin decreased albuminuria by 43.9% and eGFR by 5% . It alsoA meta-analysis showed that a reduction in albuminuria with dapagliflozin cannot be predicted by baseline characteristics or changes in most of the other metrics, such as HbA1c and body weight. Urinary albumin to creatinine ratio (UACR) reduction, although proven with SGLT2i, remains an individual characteristic with 46% patients in the study classified as ‘‘non-responders’’ . 2, concluded that canagliflozin improves renal outcomes independent of the baseline level of kidney function and a history or high risk of cardiovascular disease [Many studies analyzed both the renal and cardiovascular outcomes of SGLT2 inhibition therapy, reaching various interesting conclusions related to different sets of patients, according to the presence of different cardiovascular morbidities. An analysis of the CANVAS program, which studied the effects of canagliflozin versus a placebo on 10,142 patients with T2DM and eGFR > 30 mL/min/1.73 m disease ,104. Sim disease . In pati disease . A meta- disease .In a randomized controlled trial that included 4401 patients with T2DM and albuminuric CKD, canagliflozin reduced the risk of end-stage renal kidney disease, a doubling of creatinine level, or death from renal causes by 34% . SignifiAs shown, the studies about SGLT2i usage in DKD patients are numerous and the clinical evidence for their benefit is vast. The same cannot yet be said for the population of patients without DM; however, the existing evidence is worth a closer look.2 and ACT of 200–5000 mg/g. The DAPA-CKD concluded that regardless of the presence or absence of diabetes, the risk of a composite of a sustained decline in the estimated GFR of at least 50%, end-stage kidney disease, or death from renal or cardiovascular causes was significantly lower with dapagliflozin than with the placebo [Heerspink et al. have been one of the first and most consistent groups in discussing the possibility of SGLT2i having beneficial effects on the kidney, independently of diabetes. In line with this, one of their early studies in 2016 showed that canagliflozin slows the progression of CKD, independently of its glycemic effects . However placebo . The sci placebo .Having experienced this success in the adult population, SGLT2 inhibitors are thought to have the same effect in children; however, their use has not yet been approved due to a lack of data. Nevertheless, it is believed that “no pathophysiological clues exclude their application”  in childRelevant studies have found similar effects in regard to sex ,122 and Another important population that currently lacks evidence for the use of SGLT2i is the kidney transplant patients, since most large studies automatically do not include them due to safety concerns. The existing data, although limited, do show a modest impact of SGLT2i on glycemic control, body weight, serum uric acid levels and blood pressure, while “the frequency of reported adverse effects does not appear to exceed those found in nontransplant patients” . FurtherAll in all, the SGLT2i are slowly but surely being proven as effective and safe in all relevant populations, with no concerning evidence being forwarded as of yet.2 and ≥25 mg/mmol, respectively. Interestingly, the UKKA guidelines recommend initiation of SGLT2i in patients with eGFR 25–60 mL/min per 1.73 m2 irrespective of DM, and even in the case of albuminuria when there is a need to modify cardiovascular risk, thereby considering the powerful cardioprotective effects of SGLT2 inhibition [SGLT2i are a cornerstone of clinical guidelines and recommendations for treatment of HF, DM and now, as is the focus of this review, CKD as well. This is due to the previously discussed studies that have propelled them into worldwide use, SGLT2i recommendations, such as the United Kingdom Kidney Association (UKKA) guidelines, are subsequently incorporated in patient settings that were included in the aforementioned studies. Therefore, the eGFR and UACR cut offs for SGLT2i use in both diabetic and non-diabetic kidney disease are set to ≥25 mL/min per 1.73 mhibition . It is nhibition .For similar reasons, with regard to the patients studied in the most important trials, such as CREDENCE and DAPA-CKD, SGLT2i are currently, as previously mentioned, not recommended to kidney transplant patients, and likewise, to patients that have taken any immunological therapy in the prior 6 months. Apart from DKD, other etiologies of CKD that have been studied in large trials, and therefore included in recommendations, are as follows: ischemic nephropathy, IgA nephropathy, FSGS, chronic pyelonephritis and chronic interstitial nephritis ,127.Whereas in DM settings, SGLT2 inhibitors are usually combined with metformin , in non-Due to their pharmacokinetics, all SGLT2i are taken once daily. Being the medication with the most clinical evidence for non-diabetic CKD, 10 mg of dapagliflozin is currently the most frequently used SGLT2i  in this In conclusion, SGLT2i have already become an important part of our therapeutical arsenal versus DM, DKD and non-diabetic CKD. The latter setting is dominated by the most frequently studied dapagliflozin, but at this rate, it will soon be enhanced by the arrivals of the others. There is a similar tendency of widening the scope of indications and reducing the number of counterindications due to the unknown, which will, hopefully, and most significantly, result in the addition of non-albuminuric CKD to worldwide recommendations, and also the inclusion of the currently excluded kidney transplant patients.There have been various studies that have assessed the potential adverse effects of SGLT2i. One of the more discussed potential adverse effects of SGLT2i is the apparent risk of urinary tract infections (UTIs) and genital infections. A meta-analysis carried out in 2017 verified an increased risk for the latter, but not the former . This viIn the previous years, several case reports and observational studies associated SGLT2i with acute kidney injury (AKI), presumably through the mechanism of increased diuresis and subsequent volume depletion. Nevertheless, all the relevant studies in the meantime have not only heavily rejected that notion , but havFor many years, there has been a concern that SGLT2i might be associated with euglycemic diabetic ketoacidosis (DKA). A possible mechanism was found in their insulin-independent reduction in blood glucose and ensuing hyperglucagonemia . SimilarConsidering their principal mechanism of action, i.e., reducing blood glucose, SGLT2i have been suspected to cause hypoglycemia. However, studies have shown that the risk is increased only when SGLT2i are combined with other glucose lowering agents, such as insulin or sulfonylureas , which cSimilar to the previously discussed hypoglycemia, SGLT2i have also been hypothesized to cause hypotension, since one of the main consequences of increased glucose and sodium reabsorption in the proximal tubule is increased diuresis and possible volume depletion. All the recent studies have, to a lesser or greater extent, confirmed that hypothesis, putting the focus on the need to adjust diuretic and/or antihypertensive treatment accordingly ,135,136.Perhaps the most controversial topic regarding SGLT2i adverse effects has been their effect on bone metabolism and fractures. It is believed that increased diuresis induced by SGLT2i might disturb calcium and phosphate homeostasis, leading to bone mineral losses . HoweverSGLT2i have also been associated with a higher risk of lower limb amputation, notably canagliflozin , while nDM induces hyperglycemia and tubular growth, which enhance the amount of sodium and glucose reabsorbed by the proximal tubule, and in turn increase GFR through the physiology of TGF and tubular back pressure. Consequently, higher tubular rates of transport and oxygen consumption ensue. SGLT2i offer a myriad of direct and indirect and renoprotective effects, thus halting the progression of CKD, even in patients without DM. Particular attention was paid to transient GFR reduction, which in fact contributes to GFR preservation in the long term. Importantly, the use of mathematical modeling provided new insights that explain why renoprotective effects that arise from SGLT2 inhibition are preserved in CKD, in spite of reduced blood glucose effects. On the other hand, it was brought into question whether SGLT2i might contribute to AKI in susceptible patients, such as volume depleted or patients receiving radiocontrast agents, especially considering the hypoxic effect of SGLT2i on the outer medulla. Nevertheless, although these claims have been disproved by clinical evidence, further consideration in this regard, but also concerning euglycemic DKA, is warranted. Currently, selective SGLT1 inhibition, as well as dual inhibition of SGLT2/SGLT1 are also being explored as potential new therapeutic strategies in CKD. Furthermore, in the upcoming years, we will also have an answer as to whether SGLT2i may be valuable for patients with type 1 DM. In summary, although SGLT2i provide clinicians with an exciting new treatment option for patients with CKD, further research is needed to determine which subgroups of patients with CKD will benefit the most, and which the least, from this option.


The glutaminolysis pathway is recognized as one of the hallmarks of glioblastoma associated with tumor cell maintenance, survival, and aggressiveness. Targeting glutaminolysis emerged as a promising strategy for tumor treatments. Still, the development of glutaminase inhibitors is limited, which demands the identification of novel inhibitors for disrupting glioblastoma metabolism and its progression. Here, we report a novel library of dioxocin derivatives as glutaminase inhibitors and their pharmacological intervention for treating glioblastoma.b,f]dioxocin derivatives as glutaminase (GLS) inhibitors and their anti-GBM potential. The library consisting of seven molecules was obtained through self-condensation of 2′-hydroxyacetophenones, out of which three molecules, namely compounds 3, 5, and 6, were identified with higher binding energy values ranging between −10.2 and −9.8 kcal/mol with GLS . Pharmacological validation of these compounds also showed a higher growth inhibition effect in GBM cells than the standard drug temozolomide (TMZ). The most promising compound, 6, obeyed Lipinski’s rule of five and was identified to interact with key residues Arg307, Asp326, Lys328, Lys399, and Glu403 of GLS. This compound exhibited the best cytotoxic effect with IC50 values of 63 µM and 83 µM in LN229 and SNB19 cells, respectively. The potential activation of GLS by the best-constrained dibenzodioxocin in the tested series increased apoptosis via reactive oxygen species production in both GBM cells, and exhibited anti-migratory and anti-proliferative properties over time in both cell lines. Our results highlight the activation mechanism of a dibenzodioxocin from the structural basis and demonstrate that inhibition of glutaminolysis may facilitate the pharmacological intervention for GBM treatment.Glutamine metabolism is an important hallmark of several cancers with demonstrated antitumor activity in glioblastoma cancer cells (GBM). GBM cells regulate glutamine and use it as a major energy source for their proliferation through the glutaminolysis process. Enzymes, such as glutaminase in glutaminolysis, can be targeted by small-molecule inhibitors, thus exhibiting promising anticancer properties. The resistance to glutaminolysis demands the development of new therapeutic molecules to overcome drug resistance. Herein, we have reported a novel library of constrained methanodibenzo[ Glioblastoma multiforme (GBM), specifically grade IV astrocytoma, is one of the fast-growing and aggressive brain tumors, which starts from either the brain or the spinal cord. It shares common characteristics with other tumors, such as uncontrolled proliferation, evasion of apoptosis, invasiveness, avoidance of immune surveillance, resistance to chemotherapy, and angiogenesis ,2. GlutaSeveral drugs have been developed to inhibit different parts of the glutaminolysis pathway a. The fib,f]dioxocin skeleton found in biologically active natural products and pharmaceuticals , and further characterized their mode of interaction with GLS. The current study intends to correlate GLS inhibition and the possible downstream effects in GBM cells. The race to develop new therapeutics in this field is discussed to provide a reference for developing a novel glutaminase modulator for the treatment of cancers.b,f]dioxocins was previously described by Assoah et al. dioxocins 1–7 and TMZ, a chemotherapeutic agent for glioblastoma treatment, were dissolved in DMSO to obtain a final concentration of 100 mM, from which intermediate dilutions were prepared. For the dose-dependent cytotoxicity assay, the final concentrations of 150 μM, 100 μM, 75 μM, 50 μM, 25 μM, and 10 μM were prepared in the same culture medium. The human glioblastoma cell lines SNB19 and LN229  were used as positive (PC) and negative controls, respectively. The cytotoxicity effect of 100 µM of the top lead compound was also tested against the growth of normal cells, MEF. The Trypan blue assay allows to measure the plasma membrane integrity, which is a widely used method for determining cell viability dioxocins can serve as potent glutaminase inhibitor lead compounds. However, considering the distinct interactions observed for these three compounds, a detailed study on the molecular diversity is further required to describe the structure–activity relationships. Nevertheless, the cell growth inhibition effect and bioavailability prediction described compound 6 as a potential lead among the other tested compounds. Glutaminase plays a crucial role in the metabolism of numerous cancers, including GBM, through the glutaminolysis pathway. Glutaminolysis is one of the major altered metabolic pathways involved in tumor growth ,31,32 an6 showed a significantly high cytotoxic effect in LN229 cells, with an IC50 of ~83.52 µM, and the SNB19 cell line, with IC50 of ~63.12 µM, which is in a close range to other synthetic glutamine compounds . The HIA plays an important role in transporting drugs into the human body and is readily absorbed by the intestine dioxocins as effective anti-GBM compounds by targeting the glutaminolysis pathway.Reactive oxygen species, such as hydrogen peroxide, hydroxyl radicals, and superoxide anions, produced in living cells play a significant role in many cellular functions , includi studies . Hence, b,f]dioxocins exhibited promising glutaminolysis pathway inhibitory activity, which was validated through the increased ROS production in GBM cells. This compound could occupy the major active site of GLS, which was confirmed by molecular docking. While the anti-GBM properties of many heterocyclic classes of compounds are well-established, to the best of our knowledge, this is the first investigation reporting the anti-GBM effects of a set of synthesized methanodibenzodioxocins. Further in vivo studies using methanodibenzodioxocins would help in identifying potential chemotherapeutic agents for treating glioblastoma.The novelty of the present work was focused on developing GLS-targeted dioxocins for treating GBM. Herein, details of the synthesis of several novel dioxocin derivatives along with their potential inhibitory activities on GBM cells through the inhibition of the glutaminolysis pathway have been discussed. All compounds were elucidated by analyzing pharmacokinetics data along with the structural interaction of the same compounds with GLS. Besides, methanodibenzo[


The expression levels of SGLT2 were evaluated by immunostaining. Furthermore, the undirected graphical model was used to identify factors associated with hepatic expression levels of SGLT2. The SGLT2 expression was observed in not only the kidney, but also the liver in immunostaining  and immunoblotting. There was no significant difference in hepatic expression of SGLT2 in the stratified analysis according to age, sex, BMI, and the severity of the liver disease. In the undirected graphical model, SGLT2 directly interacted with various factors such as sex, fatty change, neutrophil-to-lymphocyte ratio, triglyceride, hemoglobin A1c, creatinine, and albumin . The expression of SGLT2 was observed in the hepatocytes of patients with chronic liver disease. The undirected graphical model demonstrated the complex interaction of hepatic expression levels of SGLT2 with gender, inflammation, renal function, and lipid/glucose/protein metabolisms.Sodium–glucose cotransporter 2 (SGLT2) occurs in the proximal renal tubule cells. We investigate the hepatic expression of SGLT2 and its related factors in patients with chronic liver disease. This is a retrospective human study. The liver tissues were biopsied from patients with chronic liver disease (The online version contains supplementary material available at 10.1007/s00795-022-00334-9. Sodium/glucose cotransporter 2 (SGLT2) is a subtype of SGLT, which mediates glucose uptake across apical cell membranes . SGLT2 oSGLT2 is also known to play an important role in fatty acid metabolism . The livThis study aimed to investigate the expression of SGLT2 in patients with chronic liver disease. We also examined the extra-renal expression of SGLT2 in the human heart, spleen, and brain tissues in humans. We further investigated the direct factors associated with the hepatic expression level of SGLT2 in patients with chronic liver disease.This is a retrospective study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected by the prior approval of the Institutional Review Board of Kurume University School of Medicine . An opt-out approach was used to obtain informed consent from the patients, and personal information was protected during data collection.The reagents were purchased from Cosmo Bio Co., Ltd.  unless otherwise indicated. Antibodies against SGLT2 were purchased from Proteintech Group, Inc. .n = 30). The kidney, heart, spleen, and brain tissues were obtained from necropsy patients with no impairment in each organ (each n = 5).The liver tissues were obtained from patients with chronic liver disease who underwent liver biopsy , red blood cell count, hemoglobin level, white blood cell count, neutrophil and lymphocyte ratio (NLR), platelet count, fasting blood glucose level, hemoglobin A1c (HbA1c) level, and serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase, gamma-glutamyl transpeptidase (GGT), alkaline phosphatase, cholinesterase, albumin, total bilirubin, prothrombin activity, total cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, blood urea nitrogen, and creatinine. We also calculated FIB-4 Index. In addition, the presence of hepatic steatosis and liver cirrhosis were evaluated by histological findings assessed by a board-certified pathologist (J.A.).Liver specimens were fixed in 10% formalin and embedded in paraffin. Consecutive 4-μm sections were stained with hematoxylin. Immunohistochemical staining was performed on paraffin-embedded tissues. Immunohistochemical staining was performed using SGLT2  and BOND-III . Proximal renal tubule cells of the kidney were used as a positive control for SGLT2.All samples were scanned at 40 × magnification using the Nano Zoomer , which is a slide scanning system . All imaWe measured the SGLT2 staining intensity by the oval region of interest. Three independent researchers measured the intensity thrice times in a tissue image with no clinical information . The mean intensity of the nine measurements was used in the analysis.We classified all patients into the younger and older groups, or low and high BMI groups according to the median age  and BMI , respectively.We classified all patients into the high and low SGLT2 groups according to the median hepatic expression level of SGLT2 .We examined the expression of SGLT2 by immunoblotting using kidney, normal liver, heart, spleen, and brain lysates. These lysates were purchased from Pro Sci inc .Immunoblotting was performed using the anti-SGLT2 antibody  as previously described .An undirected graphical model was employed to explore the complex interaction between SGLT2 and the variables in graphs as previously described . Two varU test. All statistical analyses were conducted by a biostatistician (M.K.).Data are expressed as numbers or means ± standard errors. Differences between the two groups were analyzed using the Mann–Whitney 2. Hepatitis C virus was the major etiology of chronic liver disease followed by hepatitis B virus and non-virus. The prevalence of fatty liver and liver cirrhosis was 10.0% and 36.7% of all subjects, respectively  and BMI , respectively. There was no significant difference in hepatic expression of SGLT2 in the stratified analysis according to age, sex, and BMI We classified all patients into high and low SGLT2 groups according to the median hepatic expression level of SGLT2. There was no significant difference in the serum levels of AST, ALT, and GGT between the High and Low SGLT2 groups Figs. A, B, C. We employed an undirected graphical model to reveal the direct interaction between SGLT2 and various factors. In the undirected graphical model, SGLT2 directly interacted with seven factors including sex, fatty change, triglyceride, HbA1c, NLR, creatinine, and albumin. Of these factors, SGLT2 showed the strongest interaction with sex Fig. .Fig. 6UnIn this study, we demonstrated that the expression of SGLT2 was observed in not only in the kidney, but also in the liver, heart, spleen, and brain. Of these organs, the hepatic expression of SGLT2 was the second -highest after the renal expression. In the undirected graphical model, SGLT2 directly interacted with various factors such as sex, fatty change, triglyceride, HbA1c, NLR, creatinine, and albumin.The kidney was used as a positive control for immunostaining of SGLT2, and we reported that SGLT2 was found in the proximal renal tubule cells in this study. In rats, the SGLT2 gene is detected in the outer medulla of the kidney . In addiIn this study, we first demonstrated the expression of SGLT2 in the human liver. Expression of SGLT2 in the liver was second highest after that in the kidney. SGLT2 is thought to be found only in proximal renal tubule cells . HoweverThere was no significant difference in the hepatic expression of SGLT2 in terms of patient characteristics and biochemical parameters in our study. However, in the undirected graphical model, SGLT2 directly interacted with sex, fatty change, triglycerides, HbA1c, NLR, creatinine, and albumin. Of these factors, sex interacted the most with the expression level of SGLT2. An advantage of undirected graphical model analysis is bi-directional assessments and our results suggest the following two possibilities (1) sex was associated with SGLT2 expression or (2) SGLT2 expression was associated with sex. There was no significant difference in SGLT2 expression levels between men and women. This finding indicated that possibility 1 was denied and SGLT2 expression was associated with sex (possibility 2). To verify the possibility 2, we performed a sub-analysis and all subjects were classified into the lower or higher SGLT2 groups according to the cutoff value of 164.4, which was based on the receiver operating characteristic curve. In the higher SGLT2 group, the prevalence of males was significantly higher than in the lower SGLT2 groups (Supplementary figure).Sabolic et al. reported that, in mice, the expression level of SGLT2 was male-dominant . These sMoreover, Singh AK et al. performed a meta-analysis and reported the effect of SGLT2i was different in cardiovascular outcomes by gender , 28. On We also reported that SGLT2 was found in the apical membrane of bile duct cells, cardiomyocytes, and splenic cells. Very low expression of SGLT2 was observed in the brain. We could not examine the detail of these expressions in this study. However, SGLT2 mRNA has been reported to be detected in the liver, cardiomyocytes, and spleen , 10. BilThis study has several limitations. First, this study was conducted in a single center with a small sample size. Second, all liver tissue samples were obtained from patients with chronic liver disease. Therefore, the expression of SGLT2 in normal human liver tissue remains unclear. Third, all samples were taken from Japanese individuals; therefore, racial differences in the expression of SGLT2 remain unclear. Accordingly, further study will be designed as an international multi-center study that includes a large number of samples with normal liver tissue specimens obtained by hepatic resection for metastatic liver tumors.In this study, we demonstrated that SGLT2 occurred not only in the kidney but also in the liver, heart, spleen, and brain. Expression of SGLT2 in the liver was second highest after that in the kidney. The undirected graphical model revealed the complex interaction of hepatic expression levels of SGLT2 with various factors such as gender, inflammation, renal function, and lipid/glucose/protein metabolisms.Supplementary file1 (PPTX 3060 KB)Below is the link to the electronic supplementary material.


This systematic review sought to assess the efficacy of combining either sodium hypochlorite or povidone-iodine as disinfection solutions with non-surgical treatment of periodontitis. An electronic search was conducted through PubMed, Scopus, Web of Science, CENTRAL, and Google Scholar from inception until 10 September 2022. Outcomes included clinical outcomes  and biochemical (BAPNA level) properties. A subgroup analysis was conducted according to the assessment timepoint. Ten studies reporting the use of povidone-iodine and five studies reporting the use of sodium hypochlorite were included in this review. Overall, in the meta-analysis of povidone-iodine, no significant changes were noted in any of the assessed outcomes; however, minor changes were noted in probing pocket depth and clinical attachment level at a specific timepoint. Regarding sodium hypochlorite, a significant reduction in all clinical outcomes, except for bleeding on probing, was noted. In conclusion, the use of povidone-iodine does not result in an improvement in clinical outcomes, whereas sodium hypochlorite has promising properties that result in significant improvement in probing pocket depth and clinical attachment level. However, more studies are needed to confirm these observations. Porphyromonas gingivalis  who solved their differences through discussions. Meanwhile, [K.G.L.] was consulted when an agreement could not be reached. Noteworthy, if the full text of an article was not found, the authors of that article were contacted.A pilot extraction was carried out to design the data extraction sheet using Microsoft Excel. The data extraction sheet consisted of two parts. The first part included the baseline characteristics of included studies  and patients . Noteworthy, BOP was extracted and analyzed as both a dichotomous and continuous outcome.The risk of bias of included RCTs was assessed using the updated version of the Cochrane risk of bias (RoB-II) tool that was revised in 2019. This tool assesses the quality of RCTs at the level of five domains: randomization, deviation from intended protocols, missing outcomes data, measurement of the outcomes, and reporting of study findings. Each RCT was rated as having either low, some concerns, or a high risk of bias in each domain and collectively. This process was carried out by two reviewers who had prior training in using this sheet. Their results were compared to ensure an accurate assessment of the quality of included RCTs.I2 statistic was above 50% and the p-value was <0.05. In outcomes where heterogeneity was encountered, the restricted maximum likelihood method (REML) was used; however, in the absence of heterogeneity, the inverse-variance (IV) or Mantel–Haenszel methods were used for continuous and dichotomous outcomes, respectively. A leave-one-out sensitivity analysis was carried out to determine if the reported effect estimate of each outcome was driven by a particular study. First, a meta-analysis was conducted based on the final assessment timepoints. Then, a subgroup analysis was conducted based on all of the reported assessment timepoints. The assessment of publication bias was not eligible due to the absence of the required number of studies (n = 10) in a single meta-analysis.All statistical analyses were carried out through STATA Software . The metan command was used to compare study outcomes between the intervention and comparison groups. The random-effects and fixed-effects models were selected according to the presence or absence of heterogeneity, respectively. Heterogeneity was present if the n = 8], non-RCTs [n = 3], duplicated records [n = 2], interventions combined with other therapies such as tetracycline HCl or amino acid gel [n = 2] or reporting of irrelevant outcomes  [n = 1]. The manual search yielded two additional studies   . No signI2 = 58.75%].Additionally, no significant difference between both groups was noted at 1, 3, or 6 months . HoweverI2 = 0%]  . No signUpon doing a subgroup analysis based on the assessment timepoint, no significant differences were found between both groups at 1, 3, and 6 months .I2 = 87.18%]  . No signI2 = 0%].Upon doing a subgroup analysis based on the assessment timepoint, no significant differences were found between PVP-I + NSPT and NSPT alone at 1 and 3 months . HoweverI2 = 0%]  . No signUpon doing a subgroup analysis based on the assessment timepoint, no significant differences were found between both groups at 1, 3, and 6 months .I2 = 0%]  .I2 = 0%]  . No signUpon doing a subgroup analysis based on assessment timepoint, no significant difference in the risk of developing BOP was found between both groups at 1, 3, and 6 months , which wI2 = 0%]  . No signUpon doing a subgroup analysis based on the assessment timepoint, no significant differences were found between both groups at 1 and 3 months .I2 = 0%]  . No signUpon doing a subgroup analysis based on the assessment timepoint, no significant differences were found between both groups at 1, 3, and 6 months .I2 = 67.77%]  . NotablyUpon doing a subgroup analysis based on the assessment timepoint, no significant differences were found between both groups at 1, 3, and 6 months .I2 = 85.60%]  . The leaI2 = 92.43%].No significant difference between both groups was noted at 1, 3, or 12 months . HoweverI2 = 89.38%]  . The leaI2 = 31.69%].Upon doing a subgroup analysis based on the assessment timepoint, no significant differences were found between both groups at 1, 2, 6, 9, and 12 months . HoweverI2 = 83.76%]  . The leaUpon doing a subgroup analysis based on the assessment timepoint, no significant difference in the risk of developing BOP was found between both groups at 3, 6, 9, or 12 months . NotablyI2 = 0%]  . HoweverI2 = 0%]  [SupplemI2 = 0%] .Notably, since the meta-analysis was based on only two studies, no applicable conclusions can be drawn from this finding. A subgroup analysis based on the assessment timepoint was not feasible due to the lack of relevant data.No studies compared NaOCl + NSPT to NSPT alone in terms of RHAL, PGM, and BAPNA. Of note, only one study reported the PI and BOP ; therefoThe process of reducing or eliminating the biofilm in infected periodontal tissues has been extensively studied, reporting the importance of complex inner anatomy, the dynamic relation of the microorganism, and factors related to host response . MechaniThis present systematic review and meta-analysis assessed the clinical efficacy of using sodium hypochlorite or povidone-iodine as adjunctive to non-surgical treatment of periodontitis which might prove to be beneficial to use in periodontal pockets with a depth greater than 6 mm after the non-surgical treatment. In fact, the introduction of additional approaches might lead to a better outcome of PPD and CAL with non-surgical treatment and thus avoiding surgical intervention .Sodium hypochlorite is used worldwide as a root canal irrigating agent in endodontic treatment due essentially to its effectiveness for its organic dissolution and also to its antimicrobial activity . HypochlOur meta-analysis of five RCTs revealed a statistically significant reduction in PPD in favor of sodium hypochlorite; however, the clinical significance of this finding should be carefully interpreted since the confidence interval is almost at the null value. This difference was maintained at 1, 2, 6, and 9 months, while other assessment timepoints (3 and 12 months) revealed no significant change. Notably, the findings based on data reported at 1, 2, and 9 months are based only on one study, so more studies are still warranted to confirm this observation. Sodium hypochlorite also showed a significant reduction in CAL as compared to the standalone NSPT which was observed only after 3 months of therapy. Due to the lack of a sufficient number of studies, no conclusions can be drawn on whether or not the added benefit of sodium hypochlorite can be maintained in the long term. Although it revealed no significant change in BOP, sodium hypochlorite resulted in a significant reduction in GR.p = 0.00) which could affect the reliability of this observation. Upon doing a subgroup analysis, we noted that the assessment timepoint was a significant contributor to the observed heterogeneity, and then at 12 months a statistically significant reduction in PPD was observed . However, it should be noted that this change in the effect size is low based on Cohen’s classification . This could indicate that PVP-I might not be effective in the short term, but it can result in a significant change in the long term. That being said, future, well-designed RCTs are still needed to confirm this finding.Povidone-iodine, a known antiseptic solution with broad bactericidal properties, was reported to be used in conjunction with NSPT in periodontitis patients. Although some studies have shown a beneficial effect of using PVP-I as an add-on to NSPT, other studies reported contradictory findings. In our systematic review of PVP-I, a total of 10 studies were included and analyzed, revealing a non-significant change in almost all of the assessed clinical and biochemical parameters. A previous systematic review highlighted a significant, yet minor, reduction in PPD with the adjuvant use of PVP-I to NSPT in the treatment of chronic periodontitis; however, in the subgroup analysis based on follow-up, no significant changes were noted . ControvIn our meta-analysis, a very similar observation to PPD was noted regarding CAL, where no significant change was noted in the overall meta-analysis; however, a significant reduction in CAL was documented after 6 months of therapy. Unfortunately, PVP-I did not show any resolution of inflammation or plaque control since its use did not result in any significant differences in either plaque index or bleeding on probing as compared to NSPT alone. In addition, no significant change was noted in the remaining clinical  or biochemical (BAPNA levels) outcomes.At present, 0.12% chlorhexidine (CHX) is the most acceptable chemical agent to assess mechanical debridement ,52. The Although our study provides valuable insights into the concurrent management of periodontitis either with NaOCl or PVP-I in addition to NSPT, our study has several limitations. First, the current meta-analysis does not compare the efficacy between NaOCl or PVP-I due to the lack of direct- or indirect-comparison studies which made it impossible to conduct a meta-analysis or a network meta-analysis, respectively. Second, significant heterogeneity was encountered in a number of analyses which sometimes reflected a change based in the timepoint; however, the different application modalities and concentrations of either NaOCl or PVP-I could attribute to the observed heterogeneity. Moreover, included studies recruited patients with variable classifications of periodontitis, and this could have played a role in the encountered heterogeneity. Third, we did not assess the impact of either intervention on the microbiological outcomes due to the lack of a sufficient number of studies reporting relevant data. Fourth, the majority of included RCTs had some concerns or a high risk of bias. Fifth, the confidence interval of some outcomes  was quite wide, reflecting the imprecision in the reported effect estimate. Therefore, our findings should be carefully interpreted. Additionally, more studies are warranted to determine if the effect of either intervention (NaOCl or PVP-I) would differ based on the depth of examined roots.Based on the evidence of this systematic review, the use of povidone-iodine as an adjunctive disinfectant solution for the non-surgical treatment of periodontitis does not present an additional benefit in terms of clinical outcomes; however, using sodium hypochlorite resulted in an added benefit compared to standalone non-surgical periodontal therapy. However, more studies of better quality are still needed.


Their body composition, blood pressure, and handgrip strength were assessed. A single-slice computerized tomography image at the level of the umbilicus was acquired and the CSAs of IntraMAT, muscle tissue, and subcutaneous and visceral adipose tissues (SCAT and VAT) were calculated. The %IntraMAT significantly correlated with physical characteristics, such as age, BMI, abdominal circumference, and muscle tissue CSA. Stepwise multiple regression analysis was performed, with the %IntraMAT as the dependent variable. Muscle tissue CSA and abdominal circumference were the common independent variables across groups to explain the variability of the %IntraMAT. It was also extracted %body fat and age for Group 2, age, handgrip strength, and BMI for Group 3, and smoking category for Group 4. These results suggested that muscle tissue size and abdominal circumference would be the strong predictors to explain %IntraMAT of the trunk muscle across four groups, and that age, %body fat, BMI, and SCAT, handgrip strength and smoking category were also good predictors for each group with different exercise frequency in middle-aged Japanese men.The purpose of this study was to examine how intramuscular adipose tissue (IntraMAT) can be characterized using physical and functional characteristics, muscle size, and/or adipose tissue in four different exercise frequency groups of middle-aged Japanese men. One thousand twenty-eight middle-aged men (age, 47.5 ± 8.1 years; height, 170.5 ± 5.8 cm; body mass, 67.0 ± 9.3 kg; body mass index, 21.9 ± 5.3 kg/m It is known that the regional distribution of adipose tissue plays a significant role in the development of metabolic and cardiovascular diseases –3, chronComputed tomography (CT) imaging has been frequently used to quantify the size of muscles and adipose tissue depots using the Hounsfield Unit (HU) classified threshold –23. GoodIt is obvious that body composition is closely related to the amount of physical activity performed. An increase in physical activity, in the forms of endurance and/or resistance training, is one of the most effective means of reducing IntraMAT and increasing muscle mass –27. To oThe purpose of this study was to examine how IntraMAT can be characterized using physical characteristics, muscle tissue CSA, and VAT or subcutaneous adipose tissue (SCAT) CSAs in participants with different exercise frequency in middle-aged Japanese men. We hypothesized that IntraMAT would be associated with muscle tissue CSA across groups with different exercise frequency, because there appears to be a robustness in the relationship between IntraMAT and muscle tissue CSA , 19.We performed a cross-sectional study of participants that were recruited for the Toyota Motor Corporation Physical Activity and Fitness Study  between October 2015 and January 2016. One thousand twenty-eight middle-aged men were recruited. Before the study, the procedure, purposes, risks, and benefits associated with the study were explained and the written informed consent of all the participants was obtained. If the consent was waived before the measurement, the measurement was not performed, and if after the measurement, all data of the corresponding participants were deleted. The participants comprised skilled workers, clerical workers, and technicians and their specific work activities ranged from desk work to computer-aided design work, quality control, machining, and transportation. The participants were allocated to four groups, depending on the frequency with which they exercised each week, as recorded in a questionnaire. Group 1 comprised individuals who did not habitually exercise; Groups 2 and 3 comprised individuals who exercised once a week and twice a week or more, respectively; and Group 4 comprised individuals who exercised daily. The physical and demographic characteristics of the participants are shown in Height, body mass and percentage body fat were measured using an automated body composition analyzer . Abdominal circumference was measured at the level of the umbilicus in the standing position at the end of expiration, while breathing normally. Resting blood pressure was measured using commercially available blood pressure measuring devices.Transaxial images were obtained at the level of the umbilicus at the end of expiration using low-dose CT imaging , essentially as performed previously . RepreseThe analysis in this study shows the results for the left and right muscles. Group 1 included individuals who has no exercise habit. Groups 2 and 3 were individuals with exercise habits once a week and twice a week or more, respectively. Group 4 consisted of individuals with exercise habit every day. Age of images of participants in Groups 1, 2, 3, and 4 were 47, 47, 48, and 51 years.To analyze the CT images, the HU threshold for adipose tissue was calculated as the mean of three regions-of-interest (ROIs) located in the SCAT of each participant. The HU threshold for skeletal muscle was calculated as the mean of three ROIs located in the psoas muscle, because this muscle is known to have the lowest adipose tissue content of the muscles in the trunk region . The mus2) = (muscle CSA)—(IntraMAT CSA)Muscle tissue CSA (cm%IntraMAT = (IntraMAT CSA) / (muscle CSA) x 100We also measured the CSAs of the VAT and SCAT. Image analysis was performed using the “region growing”, 2D, and morphology functions of SliceOmatic 5.0 software  by individuals who were blinded regarding the participant characteristics. The intra-class correlation coefficients for the analysis of each tissue were 0.999 for VAT, 0.938 for skeletal muscle, 0.855 for IntraMAT, and 0.999 for SCAT .Handgrip strength was measured using a pre-calibrated handgrip dynamometer , with the participant standing upright. The maximum muscle strengths of the right and left hands were measured alternately, with a short interval between tasks, and the mean maximum force of each hand was used in subsequent analyses.A self-administered questionnaire was used to quantify the participant’s smoking and drinking habits. Smoking habits were categorized as 1 for no smoking, 2 for previous smoking, and 3 for current smoking. Alcohol consumption habits were categorized as 1 for no alcohol consumption, 2 for occasional consumption, 3 for consumption on 3 days a week, and 4 for daily consumption.2 was calculated to evaluate the effect size among the groups. The Pearson product-moment correlation coefficient (r) was used to identify associations between variables. Forward stepwise multiple regression analysis was performed using variables that made a significant contribution to the variance . The dependent variable was the %IntraMAT of the trunk muscles and the independent variables were age, BMI, %body fat, abdominal circumference, diastolic blood pressure, systolic blood pressure, alcohol consumption category, smoking category, VAT and SCAT CSAs, and muscle tissue CSA and frequency of exercise , on the basis of the findings of our previous studies [All values are reported as mean and standard deviation. One-way analysis of variance (ANOVA) was used to compare data for Group 4 with data for the other three groups. The partial η studies , 20, 32 3,1024 = 13.7, P = 0.001, partial η2 = 0.039); %body fat ; SCAT .Pairwise comparisons revealed that individuals in Group 4 were significantly older than those in Groups 1 (P = 0.001) and 2 (P = 0.001), the %body fat of Group 4 was significantly lower than those of Groups 1 (P = 0.025) and 2 (P = 0.020), and the SCAT CSA of Group 4 was significantly lower than those of Groups 1 (P = 0.011) and 2 (P = 0.024).3,1024 = 3.68, P = 0.012, partial η2 = 0.011). Pairwise comparisons revealed that the muscle tissue CSA of the trunk muscles as a whole in Group 3 were significantly higher than those of Group 4 (P = 0.030).Stepwise linear regression analysis was then performed using the %IntraMAT as the dependent variable and 17 independent variables for each group . In all By excluding muscle tissue CSA and abdominal circumference from the independent variables which were found to have a strong influence on %IntraMAT and adding the frequency of exercises, another stepwise multiple linear regression analysis was performed on all participants to find factors that could better explain the %IntraMAT . Then, vThe purpose of this study was to examine how the IntraMAT can be characterized using physical characteristics, muscle tissue CSA, VAT and SCAT CSAs, and handgrip strength in participants with different exercise frequency in middle-aged Japanese men. Contrary to our expectations, the difference in the frequency of exercise did not show up clearly in the IntraMAT CSA, muscle tissue CSA, or %IntraMAT among the exercise groups . HoweverThere were significant differences in some parameters of physical characteristics among four groups. These points should be noted when considering the differences in parameters between the groups shown below. The %body fat and SCAT CSA of Group 4 were significantly lower than those of Groups 1 and 2, but not of Group 3 . This fiNo statistical processing has been performed, but we found that almost all the correlation coefficients for the relationships between %IntraMAT and VAT CSA appeared to be similar to those for the relationship between %IntraMAT and SCAT CSA in all the groups . A similOne of the most important findings of the present study was that IntraMAT and muscle tissue CSA inversely correlated in all groups . This fiWhat we should note is that there are racial difference in the IntraMAT accumulation. Gallager et al.  showed t2 = 0.425, P = 0.001); muscle tissue CSA, abdominal circumference, and %body fat for Group 2 ; muscle tissue CSA, abdominal circumference, age, handgrip strength, and BMI for Group 3 ; and muscle tissue CSA, abdominal circumference, and smoking habits for Group 4 . Among the identified independent variables, muscle tissue CSA and abdominal circumference were common for all the groups, which implies that these variables can explain the %IntraMAT of each group, and furthermore, the absolute standardized regression coefficients for the muscle tissue CSA were the highest, followed by those for abdominal circumference.Stepwise linear regression analysis for each group, using the %IntraMAT of the trunk muscles as the dependent variable, revealed relationships with muscle tissue CSA and abdominal circumference for Group 1 . In addition, the adjusted R2 values in the present study (2 = 0.425–0.593) were similar to those obtained in our previous study, as shown above, which suggests that 40%–50% of the variance in %IntraMAT may be explained by the muscle tissue CSA, irrespective of the age of the individuals. Furthermore, the same relationship between IntraMAT and muscle tissue CSA was identified in the trunk and thigh muscles, suggesting that this relationship may exist in multiple anatomic locations.The finding that muscle tissue CSA was able to at least in part explain the %IntraMAT of the trunk muscles across the groups is consistent with the finding of our previous study of 30 young and older individuals  that theThe other common independent variable that may explain the %IntraMAT was abdominal circumference. Abdominal circumference is a simple and easy method of estimating VAT CSA . AlthougSince muscle tissue CSA and abdominal circumference were found to have a strong influence on %IntraMAT in each group, stepwise multiple regression analysis was performed on all subjects without including these two factors as independent variables. As a result, two previously non-extracted variables, i.e. VAT CSA and SCAT, and four previously selected variables, i.e. age, %body fat, and BMI, were extracted. This result seemed reasonable, because both VAT CSA and SCAT were significantly correlated with %IntraMAT in all groups in 3) was significantly lower than those of former smokers (2.42 cm3) and current smokers (2.51 cm3) in their study of 3,010 middle-aged men and women. However, the difference in the IntraMAT among the three groups was very small (≤ 0.26 cm3). In the present study, there were no significant differences in IntraMAT among the four groups (Finally, the participants’ smoking habits were also found to, at least in part, explain the %IntraMAT of Group 4. Terry et al.  found thr groups , and we The limitation of this study was we asked frequency of exercise a week using a questioner, therefore, we do not have any information on intensity, duration and types of exercise. This information may be more important parameters than frequency of exercise a week. We believe that these points can be explored in future studies.In conclusion, we have determined the effect of exercise frequency on the relationships of %IntraMAT with body composition, handgrip strength and muscle tissue CSA in 1,028 middle-aged men. Stepwise multiple linear regression analysis revealed that muscle tissue CSA and abdominal circumference were independent variables that could explain the variability in the %IntraMAT of the trunk muscles. These results suggested that muscle size and abdominal circumference would be the strong predictors to explain %IntraMAT of the trunk muscle across four groups, and that age, %body fat, BMI, and SCAT, handgrip strength and smoking category were also good predictors for each group with different exercise frequency in middle-aged Japanese men.


For a few decades, animal models (AMs) in the form of best linear unbiased prediction (BLUP) have been used for the genetic evaluation of animals. An equation system is set in the order of all the effects in the model, including all the animals in the pedigree. Solving these large equation systems has been a challenge. Reduced AM (RAM) was introduced in 1980, which allowed setting up equations for parents instead of all animals. That greatly reduced the number of equations to be solved. The RAM is followed by a back-solving step, in which progenies’ breeding values are obtained conditional on parental breeding values. Initially, pedigree information was utilized to model genetic relationships between animals. With the availability of genomic information, genomic BLUP (GBLUP), single-step GBLUP (ssGBLUP), and single-step marker models were developed. Single-step methods utilize pedigree and genomic information for simultaneous genetic evaluation of genotyped and nongenotyped animals. There has been a shortage of studies on RAM development for genetic evaluation models utilizing genomic information. This study extended the concept of RAM from BLUP to the single-step methods. Using example data, three RAMs were described for ssGBLUP. The order of animal equations was reduced from the total number of animals to (1) genotyped animals and nongenotyped parents, (2) genotyped animals and nongenotyped phenotyped animals, and (3) genotyped animals and nongenotyped parents of phenotyped nongenotyped nonparents. Solutions for the remaining animals are obtained following a back-solving step. All the RAMs produced identical results to the full ssGBLUP. Advances in computational hardware have alleviated many computational limitations, but, on the other hand, the size of data is growing rapidly by the number of animals, traits, phenotypes, genotypes, and genotype density. There is an opportunity for a RAM comeback for the single-step methods to reduce the computational demands by reducing the number of equations. Reduced animal models used to reduce computational demands for best linear unbiased prediction (BLUP) are now applicable to single-step genomic BLUP. Since a few decades ago, animal genetic evaluations have been performed using “animal model” (AM) in the form of best linear unbiased prediction BLUP; , taking Worldwide, many genetic evaluation centers perform BLUP and single-step evaluations [ssGBLUP or single-step marker model (ssMM)] alongside each other, mainly for validation purposes and for qualifying their genomic data. Theoretically, genetic evaluation of animals unrelated to genotyped animals and their relatives remains intact in ssGBLUP compared with BLUP. However, minor differences are expected due to possibly different convergence behavior of BLUP and ssGBLUP equations .The concept of a reduced model  was first introduced to animal breeding by There have been tremendous advances in computational hardware, but on the other hand, data sizes used in genetic evaluations have increased considerably, mainly by larger pedigrees, more traits and phenotypes, and the incorporation of genomic data in genetic evaluations , 2016. TThe example data, code (written in the R programming language), and results of this study are publicly available .No Animal Care and Use Committee approvals were required for this study because example data were used.The sample pedigree is illustrated in The phenotypes were 5.64, 4.30, 4.32, 5.39, 7.72, and 4.36 for animals 4, 5, 9, 10, 11, and 12, respectively. Genetic and residual variances were considered to be 1 and 2, respectively.A−1) asAnimals with no genetic information flow with genotyped animals might be discarded from the analysis. Breeding values of those animals are independent of genomic information and can be obtained via BLUP. Animal 3 in the example pedigree  is a remA00 can be discarded. A33 contains nongenotyped animals that are parent, or progeny, or mate to a genotyped animal, and A44 contains nongenotyped animals that are parent, or progeny, or mate to an animal in A33. Groupingwhere genotyped animals are denoted as 2, rows and columns corresponding to A11, H−1 (the inverse of the augmented pedigree- and genomic-based additive genetic relationship matrix) is reduced frominto where andX1 and X2.It is assuming no or negligible changes in the solution of fixed effects available in The ssGBLUP MME is written asy is the vector of phenotypes, X is the incidence matrix relating y to fixed effects, Z is the incidence matrix relating y to animals, R is the diagonal matrix of residual variance(s) corresponding to y, where p) and n), where n, and columns corresponding to p. ConsideringThe RAM method of  animals . This maD, where and W, Compared with the full AM, respectively. The difference between  for Rnn . Whereass and d are sire and dam of n. Matrix B can be simplified to m), and others (n). Combining m and 2 in p, Equation where whereFollowing whereandBoth m), and nongenotyped nonphenotyped (n), and combining m and 2 in p, Equation This method  reduces 0 corresponds to the columns of Z for animals n. The above equation can be reduced towhere whereBack-solving for p  into genotyped animals and nongenotyped parents of phenotyped nongenotyped nonparents (q), and nongenotyped parents not parent to any phenotyped nongenotyped nonparent animal (r), Equation This method further reduces the RAM created by Method 1. Consider the RAM presented in Equation W corresponding to q, and where q. Back-solving where whereM2 is the observed and centered genotype matrix for genotyped animals, M1 is the imputed genotype matrix for nongenotyped animals derived by solving A11M1 = −A12M2, p). Thus, A−1 is replaced with A11 with A12 with Applying Method 1 to Equation n) is done by extending Equation Back-solving for nongenotyped nonparents (If p). Thus, A−1 is replaced with A11 with A12 with Z1 with Applying Method 2 to Equation n) is performed by solvingBack-solving for nongenotyped nonphenotyped animals (q). Thus, A−1 is replaced with A11 with A12 with Applying Method 3 to Equation n) is done according to Equation r) is done by solvingBack-solving for nongenotyped nonparents  are excluded from the model. Changing from AM to RAM, W replaces Z, and H−1, whereReducing AM to genotyped animals and nongenotyped parents Dnn, W. Solving Equation Note that even though Back-solving involves Equation and returnsNotice that for nonphenotyped animal n = {1, 2, 3, 6, 7, 8}) are excluded from the model. Changing from AM to RAM, Zp replaces Z, and H−1, whereReducing AM to genotyped animals and nongenotyped phenotyped animals Solving Equation Back-solving involves Equation r = {1, 2, 3}) are excluded from the model. Changing from AM to RAM, Z, and Reducing AM to genotyped animals and nongenotyped parents of phenotyped nongenotyped nonparents Solving Equation Back-solving for Back-solving for Z and The computational efficiency of RAM depends on the size and the computational cost of deriving A22, A33, A44, and A00, respectively. Pruning the pedigree free from animals in A00, 26,344,350 animals remained in the pedigree. There were 13,980,955 nongenotyped parents in the remaining pedigree, of which 9,041,130 were parents of nongenotyped nonparents. The 243,034 genotyped animals and the 9,041,130 nongenotyped parents of nongenotyped nonparents remained for the single-step RAM. Without pedigree pruning, 243,034 genotyped animals and 10,362,206 nongenotyped parents of nongenotyped nonparents remained in the RAM. Since the aim is to keep the back-solving procedure free from genomic information, only the equations corresponding to nongenotyped animals undergo reduction. It was possible to further reduce nongenotyped parents of nongenotyped nonparents to nongenotyped parents of phenotyped nongenotyped nonparents. However, this further reduction was omitted, so that a single A real data  example was used to study the dimension reduction by RAM. There were 32,679,354 animals in the pedigree. Considering Equation Equation  correspoBreeding values of some nongenotyped animals are not influenced and do not influence the breeding values of genotyped animals. If BLUP is performed in conjunction with single-step evaluations, those animals can be evaluated via BLUP only.This study showed that RAM used to reduce the dimension of equations for BLUP can be easily extended to single-step genetic evaluations. Numerical results were shown for ssGBLUP, and the three RAMs produced identical solutions to the full ssGBLUP. No results were presented for the ssMM. However, as the ssMM is an equivalent form of ssGBLUP , indirecB in Methods 1 and Method 3, or blocks of A−1 in Methods 2 and 3, instead of the dense blocks of H−1.Compared with RAM for BLUP, genotyped animals are present in the reduced single-step model. An efficient reduced model should be followed by an easy and fast back-solving, introducing no new information other than the conditionality of the back-solving solutions to the RAM solutions. That conditionality should be defined via sparse matrices, such as the diagonal matrix The choice among the three RAMs depends on the number of nongenotyped animals remaining together with genotyped animals in RAM. It seems that Method 2 compromises the sparseness of