This study aimed to explore the possibility of utilizing hepatitis B core protein (HBc) virus-like particles (VLPs) encapsulate doxorubicin (Dox) to reduce the adverse effect caused by its off-target and toxic side effect.in vitro release, cytotoxicity, and cellular uptake of TNBC tumor-targeting GE11-HBc VLPs was then evaluated.Here, a triple-negative breast cancer (TNBC) tumor-targeting GE11-HBc VLP was constructed through genetic engineering. The GE11 peptide, a 12-amino-acid peptide targeting epidermal growth factor receptor (EGFR), was inserted into the surface protein loops of VLPs. The Dox was loaded into HBc VLPs by a thermal-triggered encapsulation strategy. The These VLPs possessed excellent stability, DOX loading efficiency, and preferentially released drug payload at high GSH levels. The insertion of GE11 targeting peptide caused improved cellular uptake and enhanced cell viability inhibitory in EGFR high-expressed TNBC cells.Together, these results highlight DOX-loaded, EGFR-targeted VLPs as a potentially useful therapeutic choice for EGFR-overexpressing TNBC. In 2022, there will be approximately 4,820,000 and 2,370,000 new cancer cases and 3,210,000 and 640,000 cancer deaths in China and the USA, respectively . Therefoin vivo  and cultured in Luria–Bertani (LB) medium at 37°C until the OD600 reached approximately 0.6–0.8; then, 0.5mM isopropyl-β-d-thiogalactoside (IPTG) was added to the culture, and cells were grown at 26°C overnight to induce protein expression. The protein was purified with Nickel affinity chromatography  as the product description described.HBc sequence was synthesized by the company, and the GE11 peptide was inserted into the MIR region by SphI single enzyme digestion. The GE11-HBc was attached to a His tag at the end of the C-terminus to facilitate protein purification. The GE11-HBc sequence was cloned into the pET28a vector 2.2The purified protein was heated at 70°C for 20 min, then centrifuged at 10,000 rpm for 30 min to collect the supernatant. The supernatant was filtered through a 0.45-μm filter and subjected to ion exchange chromatography purification . The HBc VLPs were isolated by using sucrose density gradient centrifugation. Briefly, lower-density solutions were prepared by diluting with buffer  to yield final sucrose concentrations (vol/vol) of 55%, 45%, 35%, 25%, and 15%. Crude protein was added to the top of the gradient and then centrifuged for 2 h at 35,000 rpm at 4°C. After centrifugation, the fractions were collected and analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) images.2.3482 of each group was measured after the removal of free DOX by desalting column.The DOX-loaded HBc VLPs were prepared by a thermal-triggered encapsulation strategy . The Hg To calculate the loading capacity, 1 mg/ml DOX was diluted to 0.05, 0.1, 0.15, 0.2, 0.25, and 0.5mg/ml, and the standard curve of Dox was obtained after the measurement of the absorbance at A 482 nm.2.4in vitro release process of DOX in VLPs under GSH conditions was analyzed according to a previously reported method (The d method . In briet and Ci are the concentration of the drug in the stoste at testing time point (t) and the concentration of the drug in the discarded medium at testing time points (i) before t, respectively; Vt is the volume of the stoste; Vi is the volume of discarded medium; and Wtotal is the total drug mass in VLPs.where C2.54 ) were seeded to each well of 96-well plates. The medium in each well was removed after 24 h of incubation. Then, HBc-GE11, HBc-GE11-DOX , and free DOX (0.2 mg/ml) were suspended in Dulbecco’s modified Eagle’s medium (DMEM) and added to cells. The cells were further cultured for 12, 24, 36, or 48 h before standard CCK-8 assay testing (Dojindo).EGFR-positive MCF7 and MDA-MB-231 cells  were seeded on a cover-slide system overnight. Subsequently, the cells were treated with PBS, HBc-GE11-DOX  and free DOX (1.086 mg/ml), respectively. After 0.5, 1, and 2 h incubation, the cells were washed three times with PBS and then fixed with cold 4% paraformaldehyde (PFA) for 1.5 h. The cell nucleus was stained with 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) dye for 10 min. At last, a confocal laser scanning microscope was used to obtain the image.A total of 5×102.75 cells/well) were seeded in 12-well plates, respectively, in fresh medium containing HBc-GE11, HBc-GE11-DOX, and free DOX  for 2, 4, 6, 8, 10, and 12 h. After this, cells were collected and suspended in a cold PBS buffer, then analyzed by CytoFLEX flow cytometry .For further quantification, MCF7 and MDA-MB-231 cells , and cells were lysed and purified by Nickel affinity chromatography . The expressed protein was subjected to SDS gel electrophoresis and Western blot analysis  were treated with free DOX and HBc-GE11-DOX, and images were obtained at different time intervals. The free DOX and HBc-GE11-DOX entered the MDA-MB-231 cell line very fast. Even at a feeding time as short as 0.5 h, the accumulation of DOX in cells was clearly noted for both DOX and HBc-GE11-DOX (3.5The cytotoxicity of HBc VLPs was valued first. No significant inhibition effect was observed on cellular viability in two different cancer cell lines with indicated HBc VLPs concentration and incubation times from 12 to 48 h Figure 44Here, we constructed hybrid HBc-GE11 VLPs, which presented GE11 peptide to target EGFR+ breast cancer. We examined HBc VLPs as drug delivery carriers in a model of TNBC cancer. Modified VLPs delivered DOX to EGFR-expressing cancer tissues and exhibited a GSH-dependent drug release. Our results highlight DOX-loaded, EGFR-targeted VLPs as a potentially effective therapeutic option for EGFR-overexpressing TNBC.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.LZ and HL conceived and designed the project. LZ, TZ and HL prepared the manuscript. LT carried out the experiments and analyzed the data. CW did the materials characterization. YJ, HL and TD analyzed the data. LX and HL provided funding for this project. LZ and HL cowrote the manuscript. All authors contributed to the article and approved the submitted version.


Osteoporosis is a systemic metabolic bone disorder for which inflammatory cytokines play an important role. To develop new osteoporosis treatments, strategies for improving the microenvironment for osteoblast and osteoclast balance are needed. Tumor necrosis factor-α (TNF-α) plays an important role in the initiation and development of osteoporosis. Atsttrin is an engineered protein derived from the growth factor, progranulin (PGRN). The present study investigates whether Atsttrin affects osteoclast formation and osteoblast formation. Here we show Atsttrin inhibits TNF-α-induced osteoclastogenesis and inflammation. Further mechanistic investigation indicates Atsttrin inhibits TNF-α-induced osteoclastogenesis through the TNFR1 signaling pathway. Moreover, Atsttrin rescues TNF-α-mediated inhibition of osteoblastogenesis via the TNFR1 pathway. Importantly, the present study indicates that while Atsttrin cannot directly induce osteoblastogenesis, it can significantly enhance osteoblastogenesis through TNFR2-Akt-Erk1/2 signaling. These results suggest that Atsttrin treatment could potentially be a strategy for maintaining proper bone homeostasis by regulating the osteoclast/osteoblast balance. Additionally, these results provide new insights for other bone metabolism-related diseases. The progranulin-derived synthetic protein Atsttrin affects osteoclastogenesis and osteoblastogenesis via the TNFR signaling pathway, suggesting that this protein could potentially be used to treat inflammation-related bone diseases like osteoporosis. Osteoblasts are derived from mesenchymal precursor cells and are responsible for the deposition of new bone matrix to facilitate bone mineralization during bone reconstruction4. Osteoclasts, on the other hand, are giant multinucleated cells derived from the bone marrow monocyte lineage that play an important role in reabsorbing mineralized matrix6. Osteoporosis is defined as a systemic metabolic bone disease7. In osteoporosis, the balance between osteoclasts and osteoblasts is disturbed, resulting in increased bone resorption and/or decreased bone formation, which in turn reduces bone mass and bone microstructure destruction, eventually making bones prone to fracture8. Inflammatory cytokines play an important role in the occurrence and development of osteoporosis9. TNF-α is a member of the tumor necrosis factor-α superfamily, often at the peak of the inflammatory cascade12. This study found that the TNF-α level in women with postmenopausal osteoporosis was higher than that in women without osteoporosis13. TNF-α increases receptor activator of nuclear factor kappa-Β ligand (RANKL) secretion in osteoblasts and bone marrow stromal cells, which can further induce osteoclast formation and enhance osteoclast activity and bone resorption14. It was found that the expression of TNF-α in the peripheral blood and local bone tissue of osteoporotic mice was increased in age-related and ovariectomy-induced osteoporosis mouse models, which was positively correlated with the pathological process of osteoporosis15. At the same time, TNF-α can also inhibit the differentiation of osteoblasts and eventually lead to the destruction of normal bone metabolism balance, resulting in bone loss17.Bone matrix homeostasis is a dynamically balanced system that is maintained by a balance between bone formation by osteoblasts and bone resorption by osteoclasts19. Atsttrin is a PGRN-derived engineered protein composed of three fragments of PGRN, including half-unit granulin A, C, and F plus the linked proteins P3, P4 and P5. Atsttrin is the smallest molecule of PGRN function and still has an affinity for the TNF-α receptor20. Atsttrin and PGRN have similar physiological functions and can also bind to TNFR and inhibit TNF-α signaling pathways21. Moreover, Atsttrin showed longer half-lives and stronger therapeutic effects in previous studies compared to PGRN23. At the same time, we found that Atsttrin plays an important role in protecting early inflammatory arthritis in animal models and promoting cartilage repair21.Progranulin (PGRN) is a multifunctional and widely expressed growth factor that plays an important role in many pathophysiological processes of the human body, including anti-inflammatory processes, cartilage formation, tumorigenesis, tissue repair, and wound healing. PGRN contains seven semirepeated cysteine-rich motifs that can bind to tumor necrosis factor receptors (TNFRs) and effectively inhibit the TNFα-mediated inflammatory response, which can play a protective role in inflammatory diseasesHowever, researchers have not clearly determined whether Atsttrin plays a role in preventing the development of osteoporosis. Based on the results of previous studies, taking into account the importance of TNF-α in osteoporosis, the purpose of this study was to investigate the role of Atsttrin in regulating osteoclast and osteoblast formation as well as the potential molecular mechanism involved, providing an alternative strategy for the treatment of osteoporosis.25. To determine the role of Atsttrin in TNF-α- and/or RANKL-mediated osteoclastogenesis, we cultured BMDMs in the presence of RANKL and/or TNF-α for 7 days, followed by TRAP staining. As indicated in Fig. TNF-α is reported to play a predominant role in osteoclastogenesis26. To further determine the antagonistic effect of Atsttrin on TNF-α-mediated inflammatory responses, we cultured RAW264.7 cells and BMDMs with or without TNF-α (10 ng/ml) in the absence or presence of Atsttrin (500 ng/ml) to examine the expression of downstream inflammatory factors . As shown in Fig. TNF-α is believed to have an inflammatory catabolic effect in the pathogenesis of osteoporosis28. TNFR1 is reported to promote inflammatory effects, and TNFR2 is reported to exert protective effects21. We next sought to determine whether Atsttrin mediated this effect through TNFR1 or TNFR2. Thus, we suppressed the expression of TNFR1 using its specific siRNAs in RAW264.7 cells and BMDMs, and as indicated in Fig. TNF-α exerts its effects through TNF-α receptors, including TNFR1 and TNFR229. Therefore, to investigate whether Atsttrin rescues TNF-α-mediated inhibition of osteoblastogenesis, BMMSCs were cultured in the presence of osteoblastogenesis medium with or without TNF-α (10 ng/mL) in the absence or presence of Atsttrin (500 ng/ml) for 7 days, followed by ALP staining. As revealed in Fig. TNF-α is believed to significantly inhibit osteoblastogenesise downregulated in the presence of TNF-α. However, this phenomenon was reversed when TNFR1 was knocked down. Thus, these findings indicated that Atsttrin exhibited a protective effect by inhibiting TNF-α-mediated inhibition of osteoblastogenesis primarily through the TNFR1 pathway.Given the importance of TNFR signaling as well as the nature of Atsttrin as the binding protein of TNFRs, we next determined whether Atsttrin exhibited this protective effect through the TNFR pathway. To address this issue, we suppressed the expression of TNFR1 using specific siRNAs in MC3T3-E1 cells and BMMSCs Fig. . As illue not altered in the presence of Atsttrin  with or without PI3K/Akt-signaling inhibition using 1 µM wortmannin or Erk1/2 signaling inhibition using 1 µM U0126 for 8 h, followed by real-time PCR assay. As indicated in Fig. The above in vitro experiments confirmed the role of Atsttrin in osteoclastogenesis and osteoblastogenesis. To investigate whether Atsttrin could prevent accelerated bone loss in ovariectomy-induced mice. Mice received an intraperitoneal injection of Atsttrin or PBS weekly for 8 weeks after OVX surgery. As shown in Fig. 30. However, the underlying mechanisms involved in osteoporosis remain unclear. Under physiological conditions, bone metabolism is maintained through a delicate balance between bone formation and bone resorption. Nevertheless, this balance is disrupted in osteoporosis31. Previous reports have shown that inflammation plays an important role in the development of osteoporosis32. TNF-α is located at the top of the inflammatory cascade and plays an important role in the occurrence of osteoporosis16. TNF-α can lead to decreased activity of osteoblasts and increased number and activity of osteoclasts, resulting in bone loss33.Osteoporosis is a common degenerative disease in clinical practice. With the aging of the global population, the prevalence of osteoporosis is increasing every year, resulting in substantial bone-related morbidities and increased mortality and healthcare costs18. In TNF-α transgenic mice, TNF-α overexpression resulted in significant bone loss, which was significantly reduced by the application of exogenous PGRN34. Moreover, PGRN can mediate bone formation induced by BMP-235. Further studies have shown that PGRN can promote fracture healing under physiological conditions19. It was hypothesized that Atsttrin, as an engineered protein derived from PGRN, would regulate bone metabolism balance through a mechanism highly similar to that of PGRN. Previous studies have shown that Atsttrin has an effect on osteoclastogenesis at a concentration of 100 ng/ml36. To address the issue of Atsttrin’s concentration effect on osteoclast and osteoblast formation, we established an in vitro cell model with various concentrations. As indicated in Supplementary Fig. 10. To determine the concentration of Atsttrin for activating osteoblast formation, the present study took advantage of 200 ng/ml (11.9 nM) and 500 ng/ml (29.5 nM) Atsttrin. As illustrated in Supplementary Fig. 38. We found that TNF-α can enhance the activity of osteoclasts and promote the proliferation of osteoclasts, while Atsttrin attenuated this progress. Furthermore, we established a TNF-α-induced inflammatory response model in RAW264.7 cells to explore the role of Atsttrin in inhibiting osteoclastogenesis and to investigate the mechanism underlying this effect. We found that Atsttrin effectively prevented the TNF-α-mediated inflammatory catabolism signaling pathway, as evidenced by the significantly suppressed expression levels of IL-1β, IL-6, COX-2, and iNOS and reduced serum levels of IL-1β and IL-6 fragments. Moreover, TNF-α is involved in osteoclastogenesis mainly through activation of the NF-κB, p38, and JNK signaling pathways. The levels of p-P38, p-JNK, and p-P65 were significantly reduced by Atsttrin treatment. In addition, the NF-κB signaling pathway plays a critical role in TNF-α function. Nuclear translocation of p65 can be inhibited after treatment with Atsttrin.In osteoarthritis, inflammatory bowel disease, and psoriasis, PGRN can effectively inhibit the inflammatory signaling pathway mediated by TNF-α and reduce the expression of downstream inflammatory factors, thus inhibiting the inflammatory response mediated by TNF-α40. TNF-α induces the expression of molecules associated with inflammation in osteoclast precursors, including IL-1β, IL-6, COX-2, and iNOS, through binding to its receptor TNFR1. We also assessed the expression of biomarkers in TNF-α-induced osteoclastogenesis. In addition, TNF-α inhibits osteoblast precursor differentiation through TNFR1. The additional use of Atsttrin to block TNFR1 prominently repressed this process. Previous reports have demonstrated that TNFR2 mediates protective signaling41. We found that Atsttrin can enhance osteoblastogenesis through binding to TNFR2. Therefore, during the progression of osteoporosis, we found that Atsttrin exhibited its effect through both inhibiting TNFα/TNFR1-mediated inflammation and activating anabolic TNFR2 pathways. We found that Erk1/2 and Akt signaling are involved in osteoblastogenesis. In summary, in the presence of osteoblastogenesis medium, Atsttrin strongly activates Akt/Erk1/2 signaling. However, in normal media, Atsttrin can weakly activate the Akt/Erk1/2 signaling pathways. Furthermore, Atsttrin completely lost its effect when using specific inhibitors of Akt and Erk1/2 signaling. Consequently, we found that the Atsttrin-mediated anabolic effect in osteoblastogenesis depends on TNFR2-Akt/Erk1/2 signaling. Our study provides new evidence to improve the understanding of how TNFR functions in the regulation of bone homeostasis and the specific mechanisms involved in osteoporosis. This study identified TNFR as a crucial target for osteoporosis, thus advancing the current understanding of its mechanism of action in the regulation of bone homeostasis.TNF-α plays an important role in the pathogenesis of osteoporosis and is a major inflammatory factor. There are two binding sites of TNF-α on the cell surface, TNF receptor-1 and TNF receptor-2. The interaction between TNF-α and TNFR1 mediates inflammatory signaling43. Notably, we found that intraperitoneal injection of recombinant Atsttrin could dramatically prevent bone loss in the VOX model. In the current study, we confirmed the role of Atsttrin in osteoclastogenesis and osteoblastogenesis. Although Atsttrin is derived from PGRN, whether PGRN can prevent osteoporosis has not been reported and needs further investigation.The ovariectomy-induced mouse model is an established research method for studying the pathogenesis of osteoporosis in vivo44. Antiresorptive drugs targeting osteoclasts induce a decrease in osteoblast activity, and the ability of the bone formation-promoting drug teriparatide to promote bone formation is partially counteracted by an increase in osteoclast resorption activity45. Therefore, it is important to explore drugs and protein factors that can simultaneously enhance bone formation and inhibit bone resorption. In conclusion, this study provides new ideas for research on the mechanism underlying osteoblast-osteoclast regulation under inflammatory conditions and provides a potential therapeutic target for clinical treatment.Overall, Atsttrin enhances osteoblastogenesis and inhibits osteoclastogenesis under inflammatory conditions through at least 3 pathways: a) Atsttrin directly binds to TNFR1 and inhibits TNF-α-induced osteoclastogenesis; b) Atsttrin competitively binds to TNFR1 and rescues TNF-α-mediated inhibition of osteoblastogenesis; and c) Atsttrin synthesized with osteoblastogenesis medium, enhanced osteoblast formation and activity via TNFR2-Akt/Erk1/2 signaling Fig. . There iDulbecco’s Modified Eagle Medium (DMEM)  and α-modified Eagle’s medium (#8121119) were purchased from Gibco-BRL . Specific antibodies against TRAP (#ab191406), CTSK , and RUNX-2 (#ab236639) were purchased from Abcam ; anti-GAPDH (#AF7021), anti-COX-2 (#AF7003), anti-iNOS (#AF0199), anti-TNFR1 (#AF0282), and anti-TNFR2 (#AF0364) from Affinity Biosciences (OH.USA); lamin B1 (#13435), anti-P38 (#8690), anti-p-P38 (#4511), anti-JNK (#9252), anti-p-JNK (#9255), anti-Erk1/2 (#4695), anti-p-Erk1/2 (#8544),anti-AKT (#4691),anti-p-AKT (#4060),anti-IκBα (#4814), anti-p-IκBα (#5209), anti-p-P65 (#3033), and anti-P65 (#8242) from Cell Signaling Technology .We have complied with all relevant ethical regulations for animal use. All experimental animal procedures adhered to institutional guidelines and received approval from the Institutional Animal Care and Use Committee of Shandong University . Eight to twelve weeks-old female mice were used for experiments.2 and the culture medium was refreshed every 2 days. All cells were passaged when they reached approximately 80–90% confluence.The murine cell lines RAW264.7 and MC3T3-E1 were derived from the Cell Bank of the Chinese Academy of Sciences. STR analysis was conducted to ensure the authenticity of the cell lines. The references used for authentication included the databases of the Chinese Academy of Sciences and the American Type Culture Collection. MC3T3-E1 cells and primary marrow mesenchymal stem cells were maintained in α-MEM, while RAW264.7 cells and primary bone marrow-derived macrophages were cultured in DMEM. The medium comprised 10% FBS and 1% penicillin/streptomycin. Cells were cultured at 37 °C with 5% CO2 at 37 °C. For BMDMs, M-CSF  was added to the culture medium for 6 days, and the medium was changed on the third day, BMDMs could be successfully cultured six days later. For BMMSCs, the culture medium was replaced to remove the unadherent cells after 48 h. The medium was replaced every 3 days. The adherent cells were subcultured after becoming 80–90% confluency. Passages 1–3 cells were used in this study.Primary bone marrow-derived macrophages (BMDMs) and primary marrow mesenchymal stem cells (BMMSCs)were isolated from mice. After the mice were anesthetized and humanely euthanized by cervical dislocation, they were immersed in disinfectant alcohol for 20 min, followed by the tibia and femur being isolated, and both ends of the epiphyses were cut to the open bone medullary cavity. Flush the marrow into a 15 mL centrifuge tube using a 1 mL syringe. Use 5 mL PBS per bone. Bone marrow cells were collected and seeded in 6-well plates or cell culture dishes. The cells were cultured in a saturated humidity incubator with 5% COTranscriptional expression levels were tested by collecting RAW264.7 and MC3T3-E1 cells. TRIzol reagent  was used to extract total RNA, followed by cDNA synthesis using an RT-qPCR kit . SYBR Green PCR Master Mix  was used to perform the RT-PCR reaction. ΔΔCT was employed to calculate the target gene mRNA expression. Each experiment was replicated three times. The specific primers used for this study included tartrate-resistant acid phosphatase (TRAP), Cathepsin K, Calcitonin Receptor, alkaline phosphatase (ALP), RUNX2, Col-1, Interleukin-6 (IL-6), Interleukin-1β (IL-1β), Cyclooxygenase-2 (COX-2), and β-action. The primer sequences are listed in Table Protein samples were collected from the cell lines and primary cells. Samples containing equal amounts of total protein were separated using 10% SDS-PAGE and then subjected to protein blot analysis. Following electrophoresis, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membrane was blocked using 5% skimmed milk and subsequently incubated with a specific primary antibody. An HRP-conjugated secondary antibody was applied for 1 h. Finally, these protein bands were observed using an enhanced chemiluminescence system . Particularly, RAW264.7 cells were cultured with TNF-α (10 ng/mL) in the absence or presence of Atsttrin for different time points. Anti-p65 antibody was used for Immunoblotting analysis to test the nuclear extraction (NE) and cytoplasmic extraction (CE). RAW264.7 cells were treated with TNF-α (10 ng/mL) and different concentrations of Atsttrin. Immunoblot analysis was performed with anti-iNOS and anti-COX2 antibodies. The present study found that 500 ng/ml Atsttrin could dramatically enhance osteoblast formation in osteoblastogenesis medium and remarkably inhibit catabolism. In this case, the concentration of 500 ng/ml was used for the in vitro study to make it consistent. The molecular weight of Atsttrin is 16.94 kDa. In this study, the concentration gradient of Atsttrin was set to be 10 ng/ml (0.59 nM), 42.35 ng/ml (2.5 nM), 50 ng/ml (2.95 nM), 100 ng/ml (5.9 nM), 200 ng/ml (11.8 nM), 500 ng/ml (29.5 nM), and 1000 ng/ml (59 nM).BMDMs were inoculated into 6-well cell culture plates, and the BMDM culture medium was supplemented with Macrophage Colony-Stimulating Factor (M-CSF) (10 ng/mL)  for 6 days. Subsequently, the BMDMs were treated without or with TNF-α (10 ng/mL)  in the absence or presence of Atsttrin (500 ng/ml) for 48 h. The levels of IL-1β and IL-6 in the supernatant were assessed by utilizing the ELISA kit  following the provided guidelines from the manufacturer. Moreover, the levels of CTSK and TRAP in the mouse serum were assayed through ELISA using a commercial kit  as per the manufacturer’s guidelines.BMDMs and RAW264.7 cells were treated with TNF-α (10 ng/ml), RANKL , and Atsttrin (500 ng/ml) for 7 days. The culture medium was refreshed every 2 days.The cells were fixed in 4% paraformaldehyde for 20 min. The next day, cells were stained with a TRAP Assay Kit . All procedures strictly followed the manufacturer’s instructions. Osteoclasts were identified as TRAP-positive cells with three or more nuclei.BMMSCs were isolated by the above methods. Then, the cells were plated in a six-well plate and incubated in α-MEM, which was supplemented with 1% penicillin/streptomycin and 10% FBS. One day later, the cells were rinsed with PBS and then exposed to osteoblastogenesis medium, consisting of basal medium with 10% FBS, 10 nM dexamethasone, 5 mM β-glycerophosphate, and 50 μg/ml ascorbate-2-phosphate. The osteoblastogenesis medium was regularly replaced every three days to ensure optimal conditions for cell growth and differentiation.BMMSCs were nurtured in the osteoblastogenesis medium while being exposed to 10 ng/ml TNF-α with or without Atsttrin (500 ng/ml) for 7 days. Cells were treated with BCIP/NBT Alkaline Phosphatase Color Development Kit .Cell lysis buffer without inhibitors  was used to lyse the samples. Subsequently, the cell lysate was gathered and the protein concentration was evaluated employing the BCA assay kit . The samples were determined using the ALP assay kit . The measurement of the absorbance optical density (OD) took place at a wavelength of 405 nm using the xMark Microplate Absorbance Spectrophotometer (Bio-Rad).The cells were transfected with siTNFR1 and siTNFR2 and scrambled control siRNA (scRNAi) using lipofectamine 2000 , according to the instructions. After 24 h, the culture medium changed. Western blotting was employed to assess the effectiveness of siRNA transfection. Lysate samples were gathered and subjected to mRNA and protein analysis after varying treatments.To examine the effect of Atsttrin (500 ng/ml) on cell proliferation, Cell viability was assessed using the Cell Counting Kit-8 . The procedures were executed following the guidelines provided by the manufacturer.Female C57BL/6 mice, aged eight weeks, were anesthetized with 1% sodium pentobarbital and subjected to sham operation or bilateral ovariectomy. After 8 weeks, the mice were intraperitoneally injected with PBS or Atsttrin (2.5 mg/kg body weight) twice per week for 8 weeks. Then, the femurs were harvested for analysis.46. Further, we constructed a three-dimensional model and analyzed bone mineral density (BMD), bone volume/tissue volume (BV/TV), and trabecular number (Tb.N).The femur tissues were fixed overnight in 70% ethanol, after which they underwent scanning using an in vivo micro-CT imaging system specially designed for small animals . Scan parameters were set at 6 µm per pixel resolution, 88 mA current, and 90 kV voltage. Two hundred section planes below the growth plate of the distal metaphysis as the volume of interest (VOI) was used for reconstruction and analysist-tests were used for comparisons between two independent groups, and one-way or two-way ANOVA (when appropriate) was performed. The experiments were conducted independently at least three times with duplicate samples. Statistical significance at two sides was indicated when P < 0.05.All data are presented as the mean ± standard deviation. All statistical analyses were analyzed using the GraphPad Prism software . Student Further information on research design is available in the Peer Review FileSupplementary MaterialDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Климактерические симптомы могут нарушать ход жизни женщин на пике карьеры и семейной жизни. В настоящее время самым эффективным методом лечения этих проявлений является менопаузальная гормональная терапия (МГТ). Наличие сердечно-сосудистых и метаболических заболеваний само по себе не исключает возможность назначения МГТ с целью купирования климактерических симптомов и улучшения качества жизни. Однако нередко препятствием для использования этого вида гормональной терапии являются опасения врачей, боящихся принести пациенткам больше вреда, чем пользы. Осторожность особенно важна, когда речь идет о женщинах, страдающих сопутствующими заболеваниями. Более того, следует признать, что качественных исследований относительно безопасности МГТ при основных хронических неинфекционных заболеваниях и часто встречаемых коморбидных состояниях недостаточно. В представленном согласительном документе проведен анализ всех доступных в настоящее время данных, полученных в ходе клинических исследований различного дизайна, и создан свод критериев приемлемости назначения МГТ женщинам с сопутствующими сердечно-сосудистыми и метаболическими заболеваниями. Опираясь на представленный документ, врачи различных специальностей, консультирующие женщин в климактерии, получат доступный алгоритм, позволяющий избегать потенциально опасных ситуаций и обоснованно назначать МГТ в реальной практике. Сопредседатели: Е.В. Шляхто, Г.Т. Сухих, В.Н. Серов, И.И. Дедов, Г.П. Арутюнов, И.А. СучковОтветственный секретарь рабочей группы: Я.А. ОрловаРабочая группа: Е.Н. Андреева, С.В. Юренева, И.С. Явелов, М.И. Ярмолинская, С.В. Виллевальде, О.Р. Григорян, Е.Н. Дудинская, Е.А. Илюхин, Н.А. Козиолова, И.В. Сергиенко, А.А. Сметник, Н.И. ТапильскаяЭксперты: Н.В. Артымук, А.Г. Арутюнов, В.Е. Балан, И.И. Баранов, С.А. Бобров, Р.И. Габидуллина, Н.Ю. Григорьева, И.В. Губарева, О.В. Дженина, Ю.Э. Доброхотова, С.О. Дубровина, Е.В. Енькова, Е.И. Ермакова, С.К. Зырянов, Н.Ю. Каткова, Л.Ю. Карахалис, Т.В. Кирсанова, Т.Ю. Кузнецова, Т.А. Макаренко, Л.И. Мальцева, С.В. Мальчикова, С.В. Недогода, С.Ю. Никулина, Т.А. Обоскалова, М.М. Петрова, А.Г. Плисюк, В.И. Подзолков, Н.М. Подзолкова, А.Э. Протасова, И.В. Савельева, Е.А. Сандакова, И.В. Сахаутдинова, М.С. Селихова, Т.М. Соколова, Л.С. Сотникова, Н.В. Спиридонова, Е.И. Тарловская, И.В. Фомин, М.Б. Хамошина, А.И. Чесникова, Г.А. Чумакова, И.И. ШапошникРаспоряжением Правительства РФ от 29.12.2022 г. № 4356-р утверждена Национальная стратегия действий в интересах женщин на 2023–2030 гг. Одной из важных задач государственной политики становится сохранение здоровья женщин всех возрастов, улучшение качества жизни и увеличение периода активного долголетия ..7].Перименопауза включает период менопаузального перехода + 1 год после последней менструации.Перименопауза начинается с нарушения регулярности менструального цикла («фаза менопаузального перехода») и длится до 1 года после полного прекращения менструаций. Эта фаза репродуктивного старения может наступать в широком возрастном диапазоне (от 42 до 58 лет) и длиться до 4–8 лет . Ваз. Ваз12].Вазомоторные симптомы и другие проявления климактерического синдрома не только ухудшают качество жизни женщин и ограничивают их функциональные возможности, но и ассоциированы с повышением риска развития ИБС в 1,34 раза, риска любых ССЗ — в 1,48 раза .У 15% женщин в перименопаузе и до 80% женщин в постменопаузе отмечаются симптомы генитоуринарного менопаузального синдрома (ГУМС) или вульвовагинальной атрофии (ВВА) . У 41% жКлассификация менопаузыПо времени наступления выделяют:По причине наступления выделяют естественную и ятрогенную (в том числе хирургическую менопаузу).Показания и противопоказания к назначению МГТ определяются актуальными Клиническими рекомендациями и инструкциями к конкретным препаратам.Показания к назначению МГТ .Противопоказания к назначению МГТ .Системная МГТСистемная МГТ является наиболее эффективным методом лечения вазомоторных симптомов и других климактерических проявлений, включая ГУМС. Большинство лекарственных препаратов МГТ одобрены для профилактики постменопаузального остеопороза, за исключением ультранизкодозированных форм.В таблице 3.1 представлены зарегистрированные на территории РФ препараты для системной МГТ.Локальная МГТЛокальная терапия эстрогенами (эстриолом) используется у женщин пери- и постменопаузального периода с жалобами только на симптомы ГУМС: сухость влагалища, диспареунию или дискомфорт при половой жизни, связанные с этим состоянием.Длительные наблюдения (6–24 мес) показывают отсутствие влияния локальных эстрогенов на эндометрий, поэтому не требуется дополнительного использования прогестагенов. Локальные эстрогены не повышают риск ВТЭО, РМЖ, ССЗ, гиперплазии и рака эндометрия по данным наблюдательных исследований . В таблиОсновные принципы назначения МГТИнсулинорезистентность, дислипидемия, артериальная гипертензия и абдоминальное ожирение — основные маркеры менопаузального метаболического синдрома . По сравОжирение, особенно абдоминальное, тесно ассоциировано с МС, значительно повышает кардиометаболический риск и отражается на заболеваемости, прогнозе и продолжительности жизни больных .Ожирение является независимым фактором риска развития ВТЭО. В рандомизированном исследовании «Инициатива во имя здоровья женщин» (WHI) у женщин с ожирением (ИМТ >30 кг/м²) было отмечено 3-кратное увеличение риска ВТЭО по сравнении с женщинами с нормальным ИМТ даже в группе плацебо .При ожирении нежелательно назначать препараты, содержащие гестагены с остаточной андрогенной и глюкокортикоидной активностью, предпочтение отдается метаболически нейтральным прогестагенам . После оЧастота СД 2 типа в популяции женщин составляет: в 40–44 года — 1,2%, в 45–49 лет — 2,4%, в 50–54 года — 4,2%,в 55–59 лет — 9,4% . СвоевреПо данным метаанализа 107 исследований, МГТ снижает риск развития СД 2 типа на 30% , а при уже имеющемся СД на фоне МГТ происходит снижение уровня глюкозы натощак и HOMA-IR, а также наблюдается улучшение липидного профиля и снижение АД, наряду со снижением степени абдоминального ожирения. На фоне монотерапии эстрогенами или комбинированной МГТ у женщин с СД2 типа не было отмечено увеличение риска сердечно-сосудистой смертности .При СД 2 типа предпочтителен пероральный вид МГТ, при отсутствии противопоказаний. При назначении комбинированной МГТ важно учитывать метаболические эффекты гестагена, входящего в состав комбинированной МГТ: следует остановить выбор на прогестагенах с нейтральным воздействием на метаболические процессы .Благоприятный эффект МГТ на углеводный обмен прекращается при отмене терапии.Таким образом, МГТ может быть рассмотрена в качестве терапией менопаузальных симптомов у пациенток с СД 2 типа.Совместимость сахароснижающей терапии с МГТ, заместительной терапии левотироксином натрия (L-T4), тиреостатической и дофаминергической терапией с учетом путей введения отражена в табл. 4.1 .Ключевые положенияСчитается, что МГТ с использованием в ее составе пероральных эстрогенов повышает риск венозных тромбоэмболических осложнений (ВТЭО) — тромбоза глубоких вен (ТГВ) и тромбоэмболии легочных артерий (ТЭЛА) 33]. Одн. Одн33].Так, по данным анализа крупных баз данных QResearch и CPRD, выполненного с использованием метода «случай-контроль», назначение комбинированной МГТ КЭЭ в сочетании с МПА ассоциировалось с наиболее высоким риском ВТЭО. Для перорального эстрадиола было отмечено достоверное повышение риска ВТЭО, и этот эффект был дозозависимым. В то же время для комбинации перорального эстрадиола с дидрогестероном риск ВТЭО не увеличивался ни при циклическом, ни при монофазном комбинированном режимах МГТ вне зависимости от дозы эстрадиола. Назначение трансдермального эстрадиола не было связано с увеличением риска ВТЭО как при монотерапии, так и в составе комбинированной МГТ. Вне зависимости от ИМТ, назначение комбинации перорального эстрадиола с дидрогестероном, трансдермального эстрадиола как в монотерапии, так и в комбинации с гестагеном не было связано с увеличением риска ВТЭО. В когорте женщин, имевших в анамнезе эпизоды ВТЭО и/или получающих терапию антикоагулянтами, отмечены достоверное снижение риска ВТЭО при назначении трансдермального эстрадиола в монорежиме, а также отсутствие увеличения риска ВТЭО при комбинированном использовании трансдермального эстрадиола с гестагеном и перорального эстрадиола с дидрогестероном .По данным наблюдательных исследований, на фоне применения трансдермального эстрадиола в низких (<50 мкг/сут) и более высоких дозах в монорежиме, а также его сочетания с гестагеном в циклическом или непрерывном режимах риск ВТЭО не увеличивался 34–37]. При этом, с одной стороны, есть свидетельства, что трансдермальный путь поступления эстрогенов ассоциируется с более низким риском ВТЭО, чем его пероральный прием, с другой — есть указание на отсутствие различий –37. При [38–40]. В крупном исследовании реальной клинической практики EURAS-HRT (более 30 000 женщин) был подтвержден долгосрочный профиль безопасности дроспиренон-содержащих препаратов для МГТ в отношении ВТЭО. Риск ВТЭО на фоне МГТ с дроспиреноном был сопоставим, а риск серьезных артериальных тромбоэмболических событий (главным образом острого инфаркта миокарда и ишемического инсульта) был достоверно ниже, чем при приеме другой МГТ (детального сопоставления по составу и особенностям другой МГТ не проводилось) .В целом современная низкодозированная и ультранизкодозированная комбинированная пероральная МГТ с использованием эстрадиола представляется безопасной в отношении ВТЭО и по риску венозных тромбозов сопоставимой с трансдермальной МГТ 40]. Одн. Одн40].По данным анализа крупных баз данных QResearch и CPRD, выполненного с использованием метода «случай-контроль», не было отмечено увеличения риска ВТЭО для тиболона .Локальная терапия эстрадиолом симптомов ГУМС не приводит к увеличению риска венозных тромбозов и может использоваться у всех категорий пациенток .ЛНГ-ВМС, содержащая 52 мг микронизированного левоноргестрела, также может быть использована как компонент МГТ. По данным исследований, применение ЛНГ-ВМС не приводило к повышению риска ВТЭО 42]..42].При принятии решения о возможности и составе МГТ следует учитывать, что риск ВТЭО нельзя рассматривать отдельно от других тромботических рисков. Так что даже в случаях, когда не исключено некоторое повышение риска ВТЭО, этот эффект может нивелироваться снижением частоты артериальных тромбозов и других сердечно-сосудистых осложнений, что в итоге обеспечит нейтральное или положительное воздействие на смертность 43]44].[44].43][.[44].43]Венозные тромбозыПри остром ТГВ и/или ТЭЛА МГТ противопоказана.Большинство экспертов рекомендуют отказаться от МГТ и у пациенток с ВТЭО в анамнезе 45]46].[46].45][.[46].45]При тяжелых менопаузальных симптомах помимо локального применения эстрогенов не исключается возможность использования наименьшей эффективной дозы трансдермального эстрадиола (≤50 мкг/сут) или ультранизкодозированной  пероральной комбинированной МГТ при соответствующей антикоагулянтной терапии 37]45][[45][37][[[45][37]Имеющиеся данные не позволяют однозначно судить о риске МГТ при остром тромбозе поверхностных вен (ТПВ) и ТПВ в анамнезе . РешениеВ исследованиях по оценке риска ТГВ и/или ТЭЛА после перенесенного ТПВ не проводится разделение между тромбозом неварикозных и тромбозом варикозных поверхностных вен (варикотромбофлебитом). Варикотромбофлебит в первую очередь обусловлен наличием варикозного расширения вен, которое может быть устранено задолго до назначения МГТ.Варикотромбофлебит в анамнезе следует считать ограничением для назначения МГТ при прямом указании на ТПВ в анамнезе как на противопоказание к применению в инструкции к конкретному препарату для МГТ.Варикозное расширение венНаличие варикозного расширения вен не является противопоказанием к МГТ и не должно влиять на принятие решения о назначении МГТ. На сегодняшний день нет данных, что МГТ увеличивает риск развития тромбоза варикозно измененных вен (варикотромбофлебита). Проведение ультразвукового исследования вен нижних конечностей перед назначением МГТ не требуется.ТромбофилииДанных о безопасности МГТ при антифосфолипидном синдроме очень мало . Из-за вДанные о безопасности МГТ при бессимптомных тромбофилиях ограничены. В некоторых исследованиях установлен повышенный риск развития ВТЭО при пероральной МГТ на фоне ряда тромбофилий  49]. Одн. Одн49].Решение о возможности и составе МГТ следует принимать индивидуально с учетом сведений о наличии ранее выявленной бессимптомной тромбофилии, тяжести менопаузальных симптомов, наличия дополнительных факторов риска ВТЭО, а также указания определенных тромбофилий в перечне противопоказаний в инструкции к конкретному препарату для МГТ 40]50].[50].40][.[50].40]Семейный анамнез тромбозов (венозный или артериальный тромбоз у родственников 1 степени родства в возрасте до 50 лет) указывает на повышенный риск ВТЭО, однако не является основанием для запрета МГТ 37]51].[51].37][.[51].37]По имеющимся данным, трансдермальная МГТ не увеличивает риск ВТЭО у женщин с бессимптомной тромбофилией, однако свидетельства в пользу ее безопасности в этой клинической ситуации ограничены 39]49].[49].39][.[49].39]Ограничением для применения конкретного препарата является указание на семейный тромботический анамнез и/или наличие определенных тромбофилий как противопоказание к применению в инструкции.В настоящее время нет доказательств пользы от отмены МГТ перед хирургическими вмешательствами или при госпитализации по поводу острого нехирургического заболевания  . При повВ 1998 г. исследование HERS, первое рандомизированное плацебо-контролируемое исследование гормональной терапии (ГТ) эстрогенами и прогестином для вторичной профилактики ишемической болезни сердца (ИБС) среди женщин в постменопаузе с установленной ИБС, не выявило пользы в отношении развития сердечно-сосудистых осложнений и общей смертности при использовании ГТ. Результаты этого исследования являются аргументом против начала ГТ для вторичной профилактики ИБС .Более поздний метаанализ 19 рандомизированных контролируемых исследований с участием 40 410 женщин в постменопаузе, получавших МГТ (большинство из которых принимали перорально), не выявил значительного увеличения смертности от всех причин, смертности от ССЗ или ИМ на фоне МГТ как в рамках первичной, так и в рамках вторичной профилактики сердечно-сосудистых осложнений.Анализ подгрупп, основанный на сроках начала МГТ, показал:В настоящее время старт МГТ не рекомендован женщинам с установленным диагнозом ИБС, включая стенокардию , а ИМ явМанифестация ИБС на фоне приема МГТ, как правило, предполагает ее отмену. Хотя авторы уже упомянутого исследования HERS по его результатам заключают, что, учитывая благоприятную картину ишемических событий после нескольких лет МГТ, женщинам с ИБС, уже получающим это лечение, может быть целесообразно продолжить его . МетаанаПациенткам с инсультом в анамнезе рекомендуется избегать системной МГТ и требуется рассмотреть альтернативное (негормональное) лечение. В исследовании WHI повышенный риск ишемического инсульта был отмечен как в группе комбинированной МГТ , так и в группе монотерапии эстрогенами , независимо от исходного риска пациента 55]. В м. В м55].Неатеросклеротическая/нетромботическая ИБС чаще встречается у женщин, однако в настоящее время недостаточно данных для стратификации риска применения МГТ по подтипам заболевания. Для женщин 50–59 лет с ИМ в анамнезе без обструктивной болезни коронарных артерий, спонтанной диссекции коронарных артерий, коронарной микрососудистой дисфункции или коронарного вазоспазма требуется индивидуальный подход к назначению МГТ. Рекомендуется избегать системной МГТ при спонтанной диссекции коронарных артерий из-за предполагаемой патофизиологической связи с уровнем женских половых гормонов. Эта рекомендация исходит из того факта, что >90% пациентов со спонтанной диссекцией коронарных артерий — женщины.При симптомах ГУМС женщинам с сердечно-сосудистыми заболеваниями может применяться локальная терапия эстриолом 18]56].[56].18][.[56].18]Ключевые положенияКлинические исследования показали, что по сравнению с плацебо или отсутствием лечения МГТ может значительно повысить уровень ЛВП, а также снизить уровень ОХС, ХС-ЛНП и Лп(а) [61–63]. Следует отметить, что Лп(а) является независимым фактором риска ССЗ, и в частности повторного ишемического инсульта 65]. Ста. Ста65].В целом МГТ рассматривается как терапия, связанная с благоприятными изменениями параметров липидов как при кратковременном, так и при длительном применении у женщин в постменопаузе. Однако есть особенности, связанные с дозами препаратов и способом их доставки.Показано, что пероральная МГТ увеличивает концентрацию ТГ по сравнению с трансдермальной МГТ . УмереннВ то же время пероральная МГТ связана с положительным влиянием на уровень ХС-ЛНП, а концентрация именно этого проатерогенного фактора в наибольшей степени влияет на развитие атеросклероза и дестабилизацию атеросклеротических бляшек (АСБ).Вопрос о том, может ли МГТ в низких дозах оказывать такое же влияние на липидный профиль, как и стандартные дозы МГТ, все еще остается неясным. Одно исследование показало, что низкие дозы МГТ были связаны с более высокими уровнями ОХС и ХС-ЛНП, более низким уровнем ТГ, чем стандартные дозы . Другие Кроме того, было обнаружено, что низкие дозы эстрадиола в составе МГТ могут снижать уровень ХС-ЛВП. Эпидемиологически низкий уровень ХС-ЛПВП в плазме был связан с повышенным риском ишемических ССЗ . В совокСуществуют противоречивые данные в отношении влияния тиболона на липидный профиль. Метаанализ, проведенный в 2021 г., показал, что тиболон снижает уровни ОХС, ХС-ЛВП и ТГ. Концентрации ЛНП значительно снижаются, если прием тиболона длится ≥26 нед . В отношИмеются данные о повышенном риске ИБС у женщин, получавших комбинированную эстроген-гестагенную терапию, но не у женщин, получавших монотерапию эстрогенами . К сожалХотя результаты ряда исследований продемонстрировали положительное влияние МГТ на липидный профиль, необходимо подчеркнуть, что МГТ не рекомендуется для терапии дислипидемии и снижения риска сердечно-сосудистых заболеваний .Ключевые положенияСпецифичные для женщин факторы риска АГ и ССЗ в более позднем возрасте включают время наступления менархе, указания в анамнезе на нарушения менструального цикла и репродуктивной функции, миому матки, синдром поликистозных яичников, эндометриоз, неблагоприятные исходы беременности, преждевременную недостаточность яичников и менопаузу. Повышенный риск в течение репродуктивного периода жизни может способствовать более значительному увеличению риска ССЗ в пери- и постменопаузе [84–88].При АГ, как и при других заболеваниях, выделяют половые и гендерные различия, которые оказывают влияние на эпидемиологию, патофизиологию и клиническое ведение.В 2019 г. стандартизированная по возрасту распространенность АГ  во всем мире составила у женщин 32% . При этоТраектории АД в течение жизни у мужчин и женщин объясняются различиями механизмов регуляции АД, сочетанием половых и гендерных факторов 90]. У ж. У ж90].Выделяют следующие специфичные для женщин патофизиологические характеристики АГ :В постменопаузе у женщин наблюдается более быстрое (по сравнению с мужчинами такого же возраста) увеличение артериальной жесткости. У женщин пожилого возраста отмечается более высокая ригидность аорты, чем у мужчин, что, по-видимому, способствует развитию изолированной систолической АГ, неконтролируемой АГ, сердечной недостаточности с сохраненной фракцией выброса левого желудочка, аортальному стенозу, что чаще встречается у женщин 97]..97].Установлено, что менопауза удваивает риск развития АГ даже после поправки на возраст и индекс массы тела . Хотя МГКлючевые положенияКурение значительно увеличивает опасность артериальных сердечно-сосудистых событий и является фактором риска злокачественных новообразований.Курение не является фактором риска ВТЭО при МГТ (включая комбинированную пероральную МГТ). Несмотря на то что курение само по себе не является основанием для отказа от МГТ, в том числе комбинированными пероральными препаратами, необходимо соблюдать осторожность при назначении пероральной МГТ курильщицам, информировать их о рисках для здоровья, связанных с курением, и настаивать на прекращении курения 102][103[103102]Вклад трансдермальных и локальных форм эстрогенов в развитие ФП у женщин в период менопаузы не определен.Возможность назначения пероральной МГТ у женщин в пери- и постменопаузе с патологией клапанов определяется наличием осложнений:Показания и противопоказания к назначению МГТ определяются актуальными Клиническими рекомендациями и инструкциями к конкретным препаратам.Источники финансирования. Работа выполнена по инициативе авторов без привлечения финансированияКонфликт интересов. Авторы декларируют отсутствие явных и потенциальных конфликтов интересов, связанных с содержанием настоящей статьи.Участие авторов. Все авторы одобрили финальную версию статьи перед публикацией, выразили согласие нести ответственность за все аспекты работы, подразумевающую надлежащее изучение и решение вопросов, связанных с точностью или добросовестностью любой части работы.


This article has been retracted: Aging has completed its investigation of this paper. We found overlap between some of the transwell assay images used in Figures 5F, 6F, 6H, 7G and data published by other authors at earlier date [Figure 7D, F duplicate images published in unrelated papers [Figure 6K are presented in two unrelated papers [ier date . We alsod papers ,3, and id papers ,4. The a


The proportion of the elderly population continues to increase due to the global increase in longevity. Heart failure with preserved ejection fraction (HFpEF) is common in the elderly due to cellular aging, myocardial stiffness, and multiple comorbidities. This age group is often under-represented in clinical trials. In this narrative review, we looked into the latest evidence-based lines of management of HFpEF in this vulnerable cohort. In this narrative review, we brought the latest evidence on the treatment of HFpEf in the elderly. We searched the largest three scientific databases  using the search words  in different combinations. To date, screening for and treatment of the causes of HFpEF  and associated comorbidities  are the main line of management of HFpEF. A multidisciplinary team, including an HF specialist cardiologist, an HF nurse, a geriatrician, a dietician, a psychologist, a physiotherapist, and an occupational therapist, should manage HFpEF elderly patients. Other specialist input may be needed according to the patient's requirements. The evidence on the effective management of HFpEF in the elderly age group is scarce and controversial. Some studied non-pharmacological approaches include supervised exercise training, pulmonary artery pressure monitoring, and the interatrial shunt device . These modalities can only improve the symptoms and HF hospitalizations without robustly impacting cardiovascular (CV) death. Among the pharmacological approaches to treat HFpEF, only the sodium-glucose cotransporter 2 (SGLT-2) inhibitors proved efficacy in reducing the hard outcomes of CV death, HF hospitalizations, and urgent visits for HF when used in elderly HFpEF patients, irrespective of the presence of diabetes mellitus. Diuretics are only beneficial to alleviate the symptoms of fluid overload, with a risk of renal impairment in volume-depleted patients. The evidence on the effectiveness of other HF-specific disease-modifying agents in elderly HFpEF patients is controversial. Elderly patients have a higher risk of having side effects from HF medications due to the higher prevalence of polypharmacy, cognitive decline, and impairment of kidney and liver functions. Therefore, cautious initiation of HF treatment with a close follow-up of the blood pressure, liver functions, kidney functions, and electrolytes are of utmost importance. Population aging is recognized as one of the significant demographic trends due to developments in medical research and general improvements in public health, resulting in a larger share of the population that is 65 and up. By 2050, an estimated 426 million people will be 80 or older. This number is projected to triple from its 2020 level .Due to advances in cardiac interventional and imaging techniques and better-targeted medications, the elderly population now has a higher prevalence of cardiac comorbidities, hypertension, and other risk factors for heart failure (HF). Age-adjusted mortality rates and HF prevalence have decreased due to better treatment of cardiovascular disease in industrialized nations. On the other hand, the aging population means that HF is becoming more common -5. ThoseCommon causes of HFpEF in the elderly include CAD, hypertension, age-related degenerative valvular disease, arrhythmias such as atrial fibrillation (AF), cardiac amyloidosis, and pericardial diseases such as constrictive pericarditis . The MAGThe causes and effects of HFpEF have been the focus of numerous scientific investigations. In addition to preexisting risk factors, aging promotes myocardial remodeling and microvascular endothelial dysfunction, both of which can contribute to Left ventricular (LV) impairment and the development of HF. LV hypertrophy, chronotropic intolerance, and the acceleration of inflammatory pathways in the pathogenesis of heart failure can be triggered by risk factors such as obesity, hypertension, diabetes, and myocardial ischemia Figure  13]..13].It can be difficult to determine the pathophysiological mechanisms underlying HFpEF, especially in individuals with several risk factors, which makes it difficult to identify and optimize the proper treatment ,15. In aMethodologyWe searched the largest three scientific databases  using the MeSH terms and keywords  in different combinations. We retrieved 100 records initially and excluded 15 of them due to duplication. We then screened the remaining records and excluded another 29 records for the following reasons: 10 records due to the duplication of information, ten records where the HFpEF patients category was not analyzed, and nine records with inaccurate referencing (a reference to nowhere). We included the remaining 56 reports in a narrative review to indicate the latest evidence on managing HFpEF in the elderly population. The methodology is summarized in Figure Management of HFpEF in the elderlyThe 2021 ESC guidelines on the management of HF recommended that a multidisciplinary team should manage HF patients, including an HF specialist cardiologist, an HF nurse, a geriatrician, a dietician, a psychologist, a physiotherapist, and an occupational therapist, with other specialist input according to the patient's needs .Control of the Causes and Risk Factors of HFpEFMost HF-specific treatments have not significantly reduced the primary endpoints of CV morbidity and mortality in randomized controlled trials (RCTs). Therefore, the 2021 European Society of Cardiology (ESC) HF guidelines recommended controlling the causes and risk factors of HFpEF as the main line of treatment for this problem . The impHF-Specific TherapiesThese included non-pharmacological and pharmacological approaches.Non-pharmacological ApproachesExercise training: Even among the elderly, exercise training has been shown to enhance peak and submaximal exercise capacity, 6-minute walking distance, and overall quality of life compared to non-pharmacological treatments. Supervised exercise training (either resistance or endurance) was shown to improve exercise capacity (peak VO2) and reverse remodeling of the atrium and left ventricular diastolic dysfunction in all patients above and above the benefits gained from standard medical therapy . In the Device therapy: Hospitalizations for heart failure (including HFpEF) were significantly reduced using the wireless implantable hemodynamic monitoring system (CardioMEMS) of the pulmonary artery pressure ,25. The Pharmacological ApproachesThe main trials that studied the pharmacological treatment of HFpEF are summarized in Table DiureticsLoop diuretics: Loop diuretics can only control the symptoms of fluid overload in HFpEF patients; however, the evidence of their effect on CV hospitalizations and mortality is controversial . MonteroMineralocorticoid receptor antagonists (MRAs): Similar to loop diuretics, the evidence on the usefulness of MRAs in the elderly with HFpEF is inconsistent. Edelmann et al.  showed tThe use of diuretics in the elderly is generally associated with a higher incidence of adverse drug effects, including orthostatic hypotension, dehydration, renal impairment, and electrolyte disturbances. Clinicians should take care of regular monitoring of renal functions and electrolytes, patient education on orthostatic hypotension, and start the diuretics at low doses with slow titration to the desired doses .Beta-Blockers (BBs)Despite BBs' well-established benefit in treating HFrEF , their bVan Veldhuisen et al.  showed NThe risk of symptomatic bradycardia is higher in elderly patients on BBs. The penetrance of the lipid-soluble BBs  is more common in the elderly, resulting in central nervous side effects . Switching the patient to non-lipid-soluble BBs helps reduce the incidence of the latter .Renin-Angiotensin-Aldosterone System (RAAS) inhibitors:Angiotensin-converting enzyme inhibitors (ACEIs): Cleland et al.  suggesteAngiotensin-receptor blockers (ARBs): The CHARM-PRESERVED trial was a double-blinded placebo-controlled RCT that showed an improvement in the rate of HF hospitalization in HFpEF patients using Candesartan; however, the CV mortality in those patients did not improve . On the Yip et al.  assessedElderly patients are more susceptible to developing side effects from the use of RAAS inhibitors, including postural hypotension, kidney failure, and electrolyte derangement. Good patient counseling, regular monitoring of kidney functions and electrolytes, and slow dose titration can help mitigate these adverse effects . These sDigoxinIn the Digitalis Interaction Group (DIG) trial, 988 patients with an EF of more than 45% were randomly assigned to receive either a placebo or digoxin . DespiteSodium-Glucose Cotransporter 2 (SGLT-2) InhibitorsThe 2022 AHA/ACC/HFSA guidelines for the management of heart failure recommended using SGLT2 inhibitors (Class IIa recommendation) for treating patients with HFpEF . Empagliflozin proved its effectiveness in reducing the composite endpoint of cardiovascular death or hospitalization for HF in HFpEF patients across different age groups in the EMPEROR-Preserved RCT, irrespective of the diabetic state . The ageAnother SGLT2 inhibitor, Dapagliflozin, was assessed in two RCTs on HFpEF patients, the PRESERVED-HF  and the Bhatt et al. tested Sotagliflozin in two types of HFpEF patients with type 2 diabetes mellitus (T2DM): those with chronic kidney disease  and thoRisks of the use of SGLT2 inhibitors in the elderly includes increased genitourinary infections, hypoglycemia, volume depletion, bone fractures, and euglycemic diabetic ketosis. However, the risks are not increased in the elderly compared to other age groups, and the benefits of reduced CV death, HF hospitalizations, and renal adverse outcomes outweigh the risks .Angiotensin Receptor-Neprilysin inhibitor (ARNI)The PARAGON-HF was a large RCT that included 4822 HFpEF patients (46% aged 75 years or older). In that study, there was no significant difference between the ARNI Sacubitril-Valsartan group and the Valsartan groups in the composite primary endpoint of HF hospitalization or CV death. Nevertheless, the Sacubitril-valsartan group had a higher incidence of hypotension and a lower incidence of renal impairment and hyperkalemia than the Valsartan group .Treatment of HFpEF remains a significant challenge in managing HF patients, especially in the elderly. The management should focus on addressing the causes of HFpEF and the associated comorbidities in the affected individuals. To date, SGLT2 inhibitors are the only agents proven by randomized clinical trials to effectively reduce the hard endpoints of CV mortality and HF hospitalizations. Exercise training and loop diuretics can improve HF symptoms without a clear benefit on CV mortality and HF hospitalizations. The benefit of the other HF disease-modifying agents in HFpEF is controversial. Elderly HFpEF patients are susceptible to developing adverse effects from HF medications; therefore, very slow initiation of medications and close follow-up of the blood pressure, renal functions, liver functions, and electrolytes are mandatory. Adjustments in the prescriptions should be considered on a case-to-case basis.


The quality of recovery-15 (QoR-15) is a patient reported outcome questionnaire that measures the quality of recovery after surgery and anesthesia. The QoR-15 has been validated in many languages; Turkish version of the QoR-15 has not yet been established. The aims of this study were to translate the QoR-15 questionnaire into Turkish and to perform a full psychometric evaluation of the Turkish version.After translating the original English version of the QoR-15 scale into Turkish, the QoR-15T scale was psychometrically validated. This process included validity, reliability, responsiveness, feasibility. The QoR-15T was evaluated before the surgery and 24 h after surgery.A total of 210 patients completed the pre- and postoperative questionnaires, providing a completion rate of 93.75%. The correlation coefficient between QoR-15T score and VAS score was 0.644 on postoperative day 1 (p < 0.001). Inter item Cronbach’s alpha was 0.863. Global test-retest concordance coefficient was 0.98 (95% CI: 0.94–1.00).The QoR-15T scale is a reliable and valid instrument for evaluating postoperative quality of recovery in Turkish speaking patients. The psychometric characteristics used to assess postoperative quality of recovery were similar to those in the English version. Recovery is a multidimensional and complex process influenced by a variety of factors such as patient characteristics, surgical procedures, and anesthesia . The greMyles et al. developed the quality of recovery - 40 score (QoR - 40) in 2000, and it is now widely used . In 2013The aim of this study was to develop the Turkish version of QoR-15 (QoR-15T) by a translation and cultural adaptation process and to evaluate the validity, reliability, and responsiveness of the QoR-15T for Turkish patients who receive general anesthesia. The authors hypothesized that the QoR-15T would have as comprehensive validity, reliability, and responsiveness as the original English version.ClinicalTrials.gov . The study was conducted in accordance with the principles of the Declaration of Helsinki  vs. < 70 mm [poor]) .Reliability was measured for consistency of the QoR-15T. Reliability was assessed by internal consistency , split-half reliability, and test-retest reliability. The latter was tested between two repeated measurements in a convenience sample of 20 patients 60 min after the postoperative QoR-15T test. The floor and ceiling effect were determined by calculating if 15% of respondents had either the highest (150) or lowest possible score (0).Standardized response mean (SRM) and Cohen’s effect size were used to assess responsiveness. The SRM was calculated as the change scores divided by the SD of the change scores. The Cohen effect size was calculated as the average change scores (from preoperative to postoperative) divided by the SD at baseline. The patient recruitment rate and successful completion rate were used to assess the clinical feasibility of the QoR-15T.There is no standard approach to determine the sample size in scale validity and reliability studies. It is typically recommended that the minimum number of participants is 150 or that the scale be administered to 10–20 times the number of items on the scale ,20. To aThe measurement data were presented as mean ± SD values and categorical data as number (n) and percentage (%). The normal distribution of the continuous variables was tested using the Kolmogorov–Smirnov test. Associations were measured using Spearman rank (ρ) correlation coefficient. Internal consistency was measured using Cronbach α. Test-retest reliability was measured using the intraclass correlation coefficient. Changes from baseline were compared using the signed ranks test. The student’s t-test was used to compare data with normal distribution, and the Mann–Whitney U-test was applied to data not showing normal distribution, and the Chi-square test was used to compare categorical data. All statistical analyses were performed using SPSS version 25.0 for Windows software . The null-hypothesis was rejected if two-tailed p < 0.05.Of the 234 eligible patients, 10 refused to participate in this study, resulting in a 95.7% (224 / 234) recruitment rate. Fourteen patients were discharged within 24 h after surgery; thus, the completion rate at 24 h after surgery was 93.8% (210 / 224) . The meaTo assess for convergent validity, the correlation was evaluated between the QoR-15T and the VAS for recovery. The Spearman rho (ρ) correlation coefficient was 0.644 on the postoperative first day following surgery (p < 0.001). A strong correlation was determined between QoR-15T and VAS (correlation > 0.60). The correlation of each domain of QoR-15T with VAS was calculated. The VAS score was determined to be weakly correlated with psychological support (correlation < 0.30) and moderately correlated with other domains (correlation 0.30–0.60) (p < 0.001). The correlations of postoperative VAS and each domain of the QoR-15T are shown in A negative, moderate (ρ = −0.410), statistically significant correlation was determined between QoR-15T and surgical time (p < 0.001). A negative, moderate (ρ = −0.380), statistically significant correlation was determined between QoR-15T and PACU length of stay (p < 0.001). The QoR-15T and subscale scores were compared according to sex, ASA physical status, education level, and extent of surgery. A statistically significant difference between the scores of males and females was only seen in the subscale of psychological support . In terms of ASA physical status, a statistically significant difference was seen only in the subscale of psychological independence (p = 0.04). No statistically significant differences were seen between the variables of education levels. There was a statistically significant difference between minor-intermediate surgery and major-complex surgery in respect of the global QoR-15T scores . In the variable of grade of surgery, a statistically significant difference was observed between the minor-intermediate and major-complex scores in the psychological support subscale  and in the psychological independence subscale .Patients with a good or poor postoperative recovery, as indicated by a VAS value of ≥ 70 or < 70 mm, were compared to establish discriminant validity. The QoR-15T score was significantly different between these groups  and , (p < 0.0001).Reliability was evaluated by Cronbach α for internal consistency. Inter item Cronbach α was 0.863, and interdimension Cronbach α was 0.755. The inter item and inter dimension correlation matrices for QoR-15T at 24 h postoperatively are shown in Changes in the preoperative and postoperative QoR-15T and responsiveness are presented in This study was conducted to assess the psychometric properties of the QoR-15T and to compare these with the VAS values in adult patients undergoing elective surgery under general anesthesia. The study results revealed that QoR-15T is a valid, reliable, responsive, and easy to use instrument for the assessment of postoperative recovery in the Turkish population.To assess convergent validity, the QoR-15T for recovery was compared with the VAS values. Convergent validity showed that the correlation coefficient between the QoR-15T score and VAS exceeded the published recommendation ρ > 0.60), which was similar to the coefficient in the original study [0, which There was an inverse correlation between QoR-15T and surgery time and PACU time. These correlations were weaker than those mentioned in the original study . In otheThe discriminate validity of the QoR-15T was determined by comparing the scores of patients who had minor and intermediate surgery to those who had major and complex surgery, with the latter scoring significantly lower. Patients with a good or poor postoperative recovery, as determined by the VAS score, were compared to demonstrate discriminant validity. The VAS score was used to classify good and poor recovery, as it is a more objective assessment than the clinician’s or patient’s opinion ,9,10.Cronbach alpha and split-half reliability were used to analyze internal consistency. These two coefficients were both > 0.7, which satisfied the published recommendations. The QoR-40T scale inter item correlation coefficients ranged from 0.40 to 0.76, with item 7 having the lowest value. Although the original scale included groups of negatively correlated binary items, the QoR-15T scale did not have negative correlated binary items. The interdimensional correlation coefficients ranged from 0.59 to 0.86, with psychological support scoring the lowest and physical comfort scoring the highest. There was no negative correlation between binary subdimensions, or between binary items. These data were sufficient to conclude that the QoR-15T has adequate reliability.The Cronbach alpha value of the QoR-40T scale (0.936) is higher than the Cronbach alpha value of the QoR-15T scale (0.863). Both are higher than 0.7, indicating that QoR-15T can be used in place of QoR-40T to achieve the same reliability in a shorter time. As a result of the reliability analysis of the QoR-15T scale, as the corrected item-total correlations of all the items were greater than 0.3 (range: 0.355 to 0.714), it can be concluded that all the items adequately contribute to reliability. The results of test-retest reliability of the QoR-15T indicated that QoR-15T has an acceptable level of reliability. For the test-retest, the value of Cronbach α was 0.98, which exceeds the established criterion of 0.7 for good reliability.Cohen effect size and SRM values > 0.5 suggest a moderate intervention impact, and values > 0.8 suggest a strong intervention impact ,22. In tThe acceptability and feasibility of the QoR-15T was assessed using recruitment rate, completion rate, and time taken to complete the questionnaire. There was a high rate of participation and successful completion, and most patients were able to complete the questionnaire in less than 4 min. Since the completion times for the QoR-40T scale are not specified, it cannot be said what improvement the QoR-15T scale brings in terms of time. Less than 4 min has been shown to be a reasonable time to measure the quality of the review in clinical studies.During the implementation of the QoR-15T scale, certain difficulties were observed. When moving from question 10 to question 11, the inversion of the Likert scale caused confusion.It was also observed that, as the patient transitioned from part A to part B, the practitioner had to assist the patient by stating 0 for “worst possible scenario” and 10 for “best possible scenario”. As previously stated, this was the most negative aspect of the QoR-15T scale. In addition, the fact that there are two questions (items 11 and 12) on the scale to assess pain can create difficulty in understanding. In the current study, patients referred to “tolerable pain” for moderate pain and “terrible pain” for severe agonizing pain.There were several limitations in this study, since it was conducted in a single, tertiary level hospital in Turkey. The first limitation was that, as the study included patients undergoing elective surgeries under general anesthesia, caution was required when applying the QoR-15T to emergency surgery. Second, patients undergoing outpatient surgery, cardiac, and otolaryngological surgery were not included. Finally, patients with poor understanding of Turkish and serious preexisting medical conditions were excluded.In conclusion, the QoR-15 was translated into Turkish, and its usefulness was assessed in surgical patients who underwent various surgeries under general anesthesia. The results demonstrated that the QoR-15T is a valid, reliable, feasible, and responsive method of assessing postoperative recovery. When compared to the QoR-40T, the QoR-15T provides an equally comprehensive but less time-consuming assessment of a patient’s QoR following anesthesia and surgery. These results support the use of the QoR-15T during the perioperative period to evaluate patient recovery after anesthesia and surgery.


Literature reviews (LRs) identify, evaluate, and synthesize relevant papers to a particular research question to advance understanding and support decision-making. However, LRs, especially traditional systematic reviews, are slow, resource-intensive, and become outdated quickly.LiteRev is an advanced and enhanced version of an existing automation tool designed to assist researchers in conducting LRs through the implementation of cutting-edge technologies such as natural language processing and machine learning techniques. In this paper, we present a comprehensive explanation of LiteRev’s capabilities, its methodology, and an evaluation of its accuracy and efficiency to a manual LR, highlighting the benefits of using LiteRev.Based on the user’s query, LiteRev performs an automated search on a wide range of open-access databases and retrieves relevant metadata on the resulting papers, including abstracts or full texts when available. These abstracts (or full texts) are text processed and represented as a term frequency-inverse document frequency matrix. Using dimensionality reduction (pairwise controlled manifold approximation) and clustering  techniques, the corpus is divided into different topics described by a list of the most important keywords. The user can then select one or several topics of interest, enter additional keywords to refine its search, or provide key papers to the research question. Based on these inputs, LiteRev performs a k-nearest neighbor (k-NN) search and suggests a list of potentially interesting papers. By tagging the relevant ones, the user triggers new k-NN searches until no additional paper is suggested for screening. To assess the performance of LiteRev, we ran it in parallel to a manual LR on the burden and care for acute and early HIV infection in sub-Saharan Africa. We assessed the performance of LiteRev using true and false predictive values, recall, and work saved over sampling.LiteRev extracted, processed, and transformed text into a term frequency-inverse document frequency matrix of 631 unique papers from PubMed. The topic modeling module identified 16 topics and highlighted 2 topics of interest to the research question. Based on 18 key papers, the k-NNs module suggested 193 papers for screening out of 613 papers in total (31.5% of the whole corpus) and correctly identified 64 relevant papers out of the 87 papers found by the manual abstract screening . Compared to the manual full text screening, LiteRev identified 42 relevant papers out of the 48 papers found manually . This represents a total work saved over sampling of 56%.We presented the features and functionalities of LiteRev, an automation tool that uses natural language processing and machine learning methods to streamline and accelerate LRs and support researchers in getting quick and in-depth overviews on any topic of interest. Recently, the traditional emphasis of literature reviews (LRs) in identifying, evaluating, and synthesizing all relevant papers to a particular research question has shifted toward mapping research activity and consolidating existing knowledge . DespiteTo shorten the time to completion, automation tools have been developed to either fully automate or semiautomate one or more specific tasks involved in conducting an LR, such as screening titles and abstracts ,5, sourcIn 2020, our group of researchers started developing an automation tool for LRs  in orderTo assess the performance of LiteRev, we conducted a manual LR on the burden and care for acute and early HIV infection (AEHI) in sub-Saharan Africa using one open-access database, PubMed, and 2 subscription-based databases, Embase and Web of Science. AEHI contributes to continuous HIV transmission despite global achievements in HIV control ,13. AcutBased on the user’s query, LiteRev performs an automated search, using the corresponding APIs, on 8 different open-access databases: PubMed, PubMed Central, CORE, JSTOR, Paperity, arXiv, bioRxiv, and medRxiv. Available metadata, that is, list of authors and their affiliations, MeSH keywords, digital object identifier, title, abstract, publication date, journal provider, and URL of the PDF version of the full text paper, are retrieved and stored in a PostgreSQL database hosted on the local machine of the user. If the full text is not available as metadata, it is extracted automatically from the available PDF file, then, references, acknowledgments, and other unnecessary terms are removed, and the remaining text is checked to confirm that it still satisfies the search terms. To identify duplicate papers, the tool compares the title and abstract of papers. If duplicates are identified, the metadata of the papers are compared to check for any discrepancies. In cases where there are discrepancies, information is merged from different sources to collect as much information as possible on the same paper. Depending on the user’s needs and requirements, LiteRev can be performed on the abstract or on the full text.NLP has evolved rapidly, and, in particular, some powerful tools were developed to process text data much more efficiently. We included those features in LiteRev  are represented as a term frequency-inverse document frequency (TF-IDF) matrix, which is computed using the  package . A TF-ID package . The cor package .Optuna package [PaCMAP and HDBSCAN have several important hyperparameters that need to be determined. Table S1 in  package  and stor package , is the  package .YAKE package [If after 500 trials, the DBCV score is below 0.5, another round of 500 trials is performed, and so on until a DBCV score equal to or above 0.5 is reached. Once the values of the hyperparameters that maximize the DBCV score are determined, obtained clusters that are larger than 25% of the corpus are clustered again with the same entire procedure described above (starting from the text processing). Once each cluster is smaller than 25% of the corpus, its 10 most important words are extracted using the  package  to ensurScikit-Learn package [LiteRev allows the user to define or add papers in the corpus that are considered as being key to the research question. The key papers for the case study were proposed by one of the coauthors (IC), who is working on the topic of acute HIV infection. The papers were chosen by the coauthor from the previously identified literature based on a nonsystematic search and from the references of key review papers if they fulfilled inclusion criteria . Using the k-NN algorithm from the  package , a list The list of relevant papers from the k-NN search or a list of papers about one or more topics can then be exported in a CSV or HTML format, and their PDF retrieved and stored in a zip folder. For visualization and further exploration, a web-based 2D representation of the corpus is available in an HTML format. Every dot, colored according to the cluster it belongs to, represents a paper with the following available information: date, title, 10 most important keywords of the cluster’s topic, and the cluster number. When clicking on a paper (dot), direct access to the full text is provided using the URL. The manual LR aimed at summarizing the current evidence on the burden and care provided for AEHI in sub-Saharan Africa, to inform policy, practice, and research in the future, to address the following questions: What is the prevalence of AEHI in sub-Saharan Africa among people being tested for HIV? What models of care have been used for AEHI diagnosis and care, including treatment, partner notifications, and behavior change? What linkage to care has been reached? and What facilitators and barriers to AEHI care were identified?We searched all papers in PubMed, Embase, and Web of Science related to burden and care for AEHI in sub-Saharan Africa that were published from the inception of the databases to December 20, 2022, using the query: “(”early hiv“ OR ”primary hiv“ OR ”acute hiv“ OR ”HIV Human immuno deficiency virus“ OR (”Window period“ AND HIV)) AND ”. This query is specific to PubMed syntax and is the exact same for both the manual LR and LiteRev. Syntax-specific queries for the manual LR in Embase and Web of Science are shown in The studies were included if they described AEHI prevalence among the population tested for HIV or describe the diagnostic strategy, model of care or linkage to care for AEHI, including studies looking at perceptions and barriers among patients and staff. Only studies conducted in sub-Saharan Africa were included. We followed the Joanna Briggs Institute methodology for conducting LRs , and papTo assess the performance of LiteRev, we compared the results from the manual LR to the same review conducted using LiteRev. Relevant and not relevant papers, as identified by the manual LR during the title or abstract screening phase and the full-text screening phase, were defined as true labels. Suggested and not suggested papers by LiteRev were considered as predicted labels. Based on these figures, 2 confusion matrices were produced. Positive and negative predictive values , recall , and WSS ,34 were where true negatives are the number of nonrelevant abstracts that were correctly identified as nonrelevant by LiteRev, that is, that were not suggested by LiteRev for screening, and false negatives are the number of relevant abstracts incorrectly classified as nonrelevant by LiteRev.No ethics approval was applied as the underlying data are not subject to any approval. The data are publicly available metadata from scientific papers.Based on the search strategy described in the Methods section, we obtained 653 papers with metadata directly from PubMed and added 1 key paper given by the user that was not present in the list of retrieved papers. After removing duplicates (n=3), papers with no abstract available (n=15), those not in English (n=3), and empty abstract after text processing (n=2), 631 unique papers were transformed in a TF-IDF matrix comprised of 631 rows representing the corpus and 3136 columns representing the unique words (vocabulary), including n-grams.For the first embedding and clustering process, a DBCV score of 0.533 was obtained after the first 500 trials with the following best set of hyperparameters: PaCMAP: 310 dimensions and 18 neighbors; and HDBSCAN: minimum cluster size of 30 and minimum samples of 7. This resulted in 5 main clusters composed of, respectively, 203, 193, 169, 35, and 31 papers. The 3 largest main clusters contained more than 25% of the total number of papers in the corpus, which triggered 3 additional text processing, embedding, and clustering processes. The best set of hyperparameters for these additional processes can be found in Table S1 in In the end, the pool of 203 papers was split into 5 clusters , the pool of 193 papers into 7 clusters , and the pool of 169 papers into 2 clusters . In total, the corpus of 631 papers was divided into 16 clusters ranging from 13 to 98 papers. Using the search query described in the Methods section, 1721 records were retrieved, among which 653 were from PubMed and 1067 records from 2 subscription-based databases, namely, Embase and Web of Science. In total, 879 records were excluded after removing duplicates, empty abstracts, and papers that were not written in English. This resulted in 631 unique papers in PubMed and 211 unique papers in Embase and Web of Science. We also removed the 18 key papers from the PubMed corpus before the screening phases. In total, 613 papers in PubMed were screened at the title and abstract level, and 87 of them were relevant to the research question. After the full-text screening phase on these 87 relevant papers, we found 48 papers to be relevant to the manual LR.Out of the 211 unique papers from Embase and Web of Science, 46 papers were found relevant to the research question after the title or abstract screening phase , and 19 after the full-text screening phase  provided a list of 18 key papers. With these 18 key papers, we performed a k-NN search on the corpus, embedded into 310 dimensions, with k=18, the number of the nearest neighbors for PaCMAP that maximized the DBCV score of the first clustering process. The first k-NN search suggested 110 papers, including 45 of the relevant papers identified by the manual LR title or abstract screening (precision of 41%). Based on these 45 relevant papers, the second k-NN iteration suggested 26 additional papers out of which 8 were confirmed as relevant (precision of 31%). The third iteration found 9 more relevant papers out of 38 papers suggested (precision of 24%). The fourth and last iteration suggested 19 papers out of which 1 was relevant (precision of 5%).In total, 193 papers out of the 613 papers were suggested by LiteRev. Suggested papers included 64 of the 87 papers identified as relevant during the title or abstract screening of the manual LR. The 64 relevant papers found by LiteRev belonged essentially to 2 topics (30 relevant papers in one and 14 relevant papers in the other). The topic that contained 30 relevant papers had 87 papers in total and covered early diagnosis, care seeking, and interventions during the acute HIV infection stage . The topic that contained 14 relevant papers had 82 papers and covered the detection of AEHI by antibody assays and incidence estimate . Screening 53 additional papers (those not suggested by the k-NN search) from these 2 topics would allow the user to identify 3 additional relevant papers.After the full-text screening phase of the manual LR, 48 out of the 87 relevant papers from the title and abstract screening phase were deemed relevant to the research question. The 64 papers suggested by LiteRev (based on abstracts only) included 42 out of the 48 papers confirmed as relevant after the full-text screening phase of the manual LR. The processing time represents the overall computation time taken by LiteRev to complete the entire process of metadata retrieval, processing, clustering, and neighbor search. It does not include the time that the user took to check the relevance of the suggested papers. The percentage of time saved by the user is expressed by the WSS metric.It took 5 minutes for LiteRev to retrieve the metadata of the 653 papers and text process the remaining 631 abstracts and transform it into a TF-IDF matrix. Each trial of the optimization process with a specific set of hyperparameters required on average 1 minute of computation. With 3000 trials in total  run sequentially, this led to an additional 50 hours, that is, roughly 2 days, to complete the entire optimization process. This computation time can be substantially reduced by running the trials in parallel. Finally, the nearest neighbors are obtained almost instantaneously.We presented LiteRev, an automation tool that uses NLP and ML methods to support researchers in different steps of a manual LR. The identification of papers to be included in an LR is a critical and time-intensive process, with the majority of time spent in screening thousands of papers for relevance. By combining text processing, literature mapping, topic modeling, and similarity-based search, LiteRev provides a fast and efficient way to remove duplicates, select papers from specific languages, visualize the corpus on a 2D map, identify the different topics covered when addressing the research question, and suggest a list of potentially relevant papers to the user based on their input .Preliminary usage of LiteRev showed that it significantly reduced the researcher’s workload and overall time required to perform an LR. Compared to a manual LR, LiteRev correctly identified 87.5% of the 48 relevant papers , by screening only 31.5% (193/631 papers) of the whole corpus, which corresponds to a total WSS of 56% at the end of the full-text screening phase. In addition, the actual time spent on running LiteRev and retrieving the results was relatively short, and the user was free in the meantime to focus on other work. The text processing and the nearest neighbors search took no more than 5 minutes of computation for 631 papers.With its topic modeling capability, LiteRev aims at summarizing current evidence on a specific research question to inform policy, practice, and research. For our use case, LiteRev identified 5 main topics and 16 different topics related to AEHI in sub-Saharan Africa, allowing the researcher to have an overview of the different perspectives related to this research question. Finding 61 out of the 105 relevant papers after the title and abstract screening phase (including the key papers) in only 2 topics validates the quality of the clustering.LiteRev is currently limited to open-access databases that provide free APIs to abstract or full-text papers. Databases often used for LRs, such as Embase or Web of Science do not provide API access, require a subscription for accessing full-text papers, or do not allow for text mining and ML analysis. Hence, 19 relevant papers identified in Embase or Web of Science were not available to LiteRev. In addition, when performed on full texts, LiteRev currently works on digitally generated PDFs but not on image-only (scanned) PDFs.Another limitation concerns the possibility of sharing the list of potentially relevant papers with other users or reviewers. LiteRev does not offer this functionality yet; hence, double screening of papers and comparison of results are not possible at the moment. To overcome this limitation, the user has the option to export their list of papers into a CSV format, which can be uploaded on Rayyan or other similar software for systematic reviews.As of today, LiteRev is still intended to complement rather than replace full systematic reviews. Finally, by January 2023, no public web-based user interface is available yet.The systematic review tool  maintainLiteRev is developed in an iterative way with continuous integration of feedbacks from users, and its modules can easily be updated or replaced depending on the needs of the users and the technical evolutions. We are further developing LiteRev by proposing a web application with a user-friendly interface and by adding more functionality in order to better automate the different stages of an LR. We are also planning to implement a living review  by retriWe presented LiteRev, an automation tool that uses NLP and ML techniques to support, facilitate, and accelerate the conduction of LRs providing aid and automation to different steps involved in this process. Its different modules  can easily be updated or replaced depending on the needs of the users and the technical evolutions. As more papers are published every year, LiteRev not only has the potential to simplify and accelerate LRs, but it also has the capability of helping the researcher get a quick and in-depth overview of any topic of interest.


Plants will celebrate its 10th anniversary. To mark this significant milestone and celebrate the achievements made throughout the years, we intend to publish a Special Issue entitled “10th Anniversary of Plants—Recent Advances and Perspectives”. In the past decade, the continuous support of the authors, editors, and reviewers, as well as the readers, has resulted in noteworthy success and the achievement of a common goal, as well as the sustained reputation of Plants in the world of science. In parallel with the development of our journal, great success has also been achieved across the field of plant science itself, from the molecular to the ecosystem level, and many new findings are based on new methodological approaches. Apart from the fact that this Special Issue will serve as a celebration of the anniversary, it should also serve as a guide for discoveries in plant science and thus for the development of the journal.Published for the first time in 2012, Plants. “Feature papers represent the most advanced research with significant potential for high impact in the field. A Feature Paper should be a substantial original Article that involves several techniques or approaches, provides an outlook for future research directions and describes possible research applications. Feature papers are submitted upon individual invitation or recommendation by the scientific editors and must receive positive feedback from the reviewers.” The number of papers with this status in relation to the total number of papers indicates the enviable scientific quality and impact of this Special Issue. The Plants editorial team practices the “Editor’s Choice” option. “Editor’s Choice articles are based on recommendations by the scientific editors of MDPI journals from around the world. Editors select a small number of articles recently published in the journal that they believe will be particularly interesting to readers, or important in the respective research area. The aim is to provide a snapshot of some of the most exciting work published in the various research areas of the journal.” This status was deservedly awarded to four papers, two articles [This anniversary Special Issue contains 101 papers, the majority comprising articles 77 papers), followed by reviews 20 papers), communications 3 papers), and one protocol. The number of citations for this 10th anniversary Special Issue papers should be emphasized on this occasion; in the first year, this value has already reached almost 400, thereby confirming the importance and scientific impact of this collection as well as predicting an enviable future. Over 600 authors from all over the world contributed to the published papers of this Special Issue. Considering the satisfactory quality and diversity of the submissions, seven papers received the “featured” status, including four articles [0 papers, papers, 7 papers,articles , one revarticles , as wellarticles .Plants, no specific topic was defined, but all potential papers which were within the aims and scope of Plants were considered, and, based on that, the Special Issue was entitled “10th Anniversary of Plants—Recent Advances and Further Perspectives”. The published papers are characterized by great diversity concerning the topics in plant sciences, reflecting recent developments and the main trends, particularly in plant molecular biology and physiology, genetics, and phytochemistry. Though a complex task, based on the main topic, objects of research, as well as the contribution of the results, it was possible to categorize the papers into several groups. One group of papers is dedicated to molecular biology and the physiology of plants, applying modern molecular methodological approaches, cell biology, microbiology, etc., where molecular and physiological processes, interactions, stress resistance, etc. are elucidated. This group includes articles [During the establishment of the 10th anniversary Special Issue of articles ,24,25,26articles ,28, and articles ,32,33,34articles ,47,48,49articles ,51. The articles ,54,55,56articles . A similarticles ,59,60,61articles . The divarticles ,92,93,94articles , and revarticles ,100,101,Plants and, at the same time, serves as a general overview of the achievements of plant sciences in the current period. I would like to take this opportunity to thank all the authors on behalf of the editorial office, especially for their interest in participating and willingness to share their experiences in science, as well as for the high-quality contributions submitted. We would like to thank the numerous reviewers for their valuable comments, which contributed to the quality of the published articles and thus to the overall quality of the Special Issue.The abovementioned information illustrates the twofold significance of this Special Issue. It is a culmination of ten years of efforts to improve the quality of Plants editorial office, especially Ms. Sumi Sun, for their collaboration and guidance during the initiation, review, and editing process of the issue.In terms of gratitude, it is imperative to note that the realization of the Special Issue was only possible thanks to the cooperation and dedication of the Special Issue editorial team—Prof. Dr. Milan Stanković , Prof. Dr. Paula Baptista , and Prof. Dr. Petronia Carillo . I would also like to thank the Plants.It has been my great pleasure to invest time and effort in editing this special issue, as well as to contribute over the past decade in the positions of assistant and associate editor of