Nucleotide excision repair (NER) is the most versatile DNA repair pathway that removes a wide variety of DNA lesions caused by different types of physical and chemical agents, such as ultraviolet radiation (UV), environmental carcinogen benzo[a]pyrene and anti-cancer drug carboplatin. The mammalian NER utilizes more than 30 proteins, in a multi-step process that begins with the lesion recognition within seconds of DNA damage to completion of repair after few hours to several days. The core proteins and their biochemical reactions are known from in vitro DNA repair assays using purified proteins, but challenge was to understand the dynamics of their rapid recruitment and departure from the lesion site and their coordination with other proteins and post-translational modifications to execute the sequential steps of repair. Here, we provide a brief overview of various techniques developed by different groups over last 20 years to overcome these challenges. However, more work is needed for a comprehensive knowledge of all aspects of mammalian NER. With this aim, here we provide detailed protocols of three simple yet innovative methods developed by many teams that range from local UVC irradiation to in situ extraction and sub-cellular fractionation that will permit study of endogenous as well as exogenous NER proteins in any cellular model. These methods do not require unique reagents or specialized instruments, and will allow many more laboratories to explore this repair pathway in different models. These techniques would reveal intracellular movement of these proteins to the DNA lesion site, their interactions with other proteins during repair and the effect of post-translational modifications on their functions. We also describe how these methods led us to identify hitherto unexpected role of poly(ADP-ribose) polymerase-1 (PARP1) in NER. Collectively these three simple techniques can provide an initial assessment of the functions of known and unknown proteins in the core or auxiliary events associated with mammalian NER. The results from these techniques could serve as a solid foundation and a justification for more detailed studies in NER using specialized reagents and more sophisticated tools. They can also be suitably modified to study other cellular processes beyond DNA repair. The nucleotide excision repair (NER) is the most versatile DNA repair pathway that eliminates a wide variety of DNA lesions caused by different types of physical and chemical agents, such as ultraviolet radiation (UV), environmental carcinogen benzo[a]pyrene and anti-cancer drug carboplatin. It is the only repair pathway in mammalian cell that removes UV-induced DNA damage, such as cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone (6-4PP) photoproducts . The pho+) and transfers the ADP-ribose moieties to poly(ADP-ribosyl)ate or PARylate itself and DDB2 as well as other acceptors proteins (The Xeroderma pigmentosum C (XPC) protein starts the GGR sub-pathway of NER by recognizing the distortion of the DNA double helix and binding to the unpaired nucleotides facing the damaged nucleotide. Its arrival at the damage site is a prerequisite for the recruitment of downstream proteins and the repair of the lesion . In the proteins . DDB2 acproteins  to modifproteins . The TCRproteins . Once thproteins .The progress in understanding mammalian NER was relatively slower than other DNA repair pathways, largely because of a conceptual and some technical hurdles. In the early stage, the specific biochemical reactions carried out by a majority of the mammalian NER proteins were identified using the in vitro DNA repair assays with purified proteins and from their equivalent proteins in bacteria and yeast . HoweverA series of innovative methods developed over the last two decades by many groups allowed a rapid gain in our understanding of mammalian NER and challenged the repairosome concept. The development of green fluorescent protein (GFP) technology and photobleaching procedures permitted the visualization and quantification of the mobility of GFP-tagged NER factors . Based oThe most important breakthrough in understanding mammalian NER came with the simultaneous development of local irradiation technique by two independent groups . In thisDespite its numerous advantages, including the low cost and use of basic equipment, the local irradiation technique needed optimization for the visualization of different repair proteins at damage site in different cell lines . For insThus, the local irradiation techniques and its improvements have revealed roles of many proteins in mammalian NER, including the unsuspected implication of the abundant protein PARP1 . HoweverDespite the tremendous progress made in the study of mammalian NER, there is need for more studies on multiple fronts in NER. The studies on NER of chemotherapeutic drug-induced DNA damage could reveal clinically exploitable knowledge to improve therapeutic efficacy of these drugs. There could be many other unanticipated proteins, like PARP1, playing different auxiliary roles in controlling the functions of core NER proteins. There are significant gaps in our knowledge of early steps of mammalian TCR sub-pathway, such as roles of other transcription initiation and elongation factors who happen to be present in the vicinity of stalled RNA polymerase II. Lastly, we have just begun to understand TCR of nucleolar DNA at stalled RNA polymerase I, but more studies could reveal an attractive target to control cancer cells which are highly dependent on rDNA transcription to meet demands of fast-growing cells. To stimulate more studies in NER, there is a need for widespread accessibility to techniques that will allow many more laboratories to explore this repair pathway in different models. In this context, some of the leading techniques in the field, such as live cell microscopy imaging using fluorescent tagged proteins  or UVC lTherefore, here we describe detailed protocols for three relatively simple yet powerful techniques: local irradiation, in situ extraction and subcellular fractionation. These techniques use reagents and tools that are readily available in most laboratories and will allow identification of potential role of both endogenous or exogenous tagged proteins in mammalian NER in most cellular models. These techniques could be readily modified to study NER after treatment with other DNA damaging agents beyond UVC or to specifically study TCR. In addition, the cellular fractionation protocol provides an enriched nucleoplasmic or chromatin-bound protein fraction that can be useful for many downstream applications, such as immunoprecipitation and proteomics to identify the components of repair complexes or partners of the target proteins. Collectively these three simple techniques can provide an initial assessment of the functions of known and unknown proteins in the core or auxiliary events associated with the efficiency of mammalian NER. The results from these techniques could serve as a solid foundation and a justification for more detailed studies in NER using specialized reagents and more sophisticated tools, as required.•Human skin fibroblasts (GM00637-Coriell Institute) or any other cell line of interest.•Appropriate medium for the cell culture, e.g., Minimum essential medium (αMEM) .•Penicillin-streptomycin solution .•Bovine growth serum .•Solution of 0.25% Trypsin and 2.12 mM EDTA .•2HPO4) , and 20 mM potassium dihydrogen phosphate (KH2PO4)  in distillated water. Do not adjust the pH. Store at RT.1X Phosphate-buffer saline (PBS), pH 7.4. Prepare 10X buffer containing 1.37 M sodium chloride (NaCl) , 27 mM potassium chloride (KCl) , 100 mM disodium hydrogen phosphate (Na•Trypan Blue Stain (0.4%) .•PJ34 hydrochloride, PARP1 inhibitor . Prepare a 30 mM stock solution (10 mg/mL) in distillated water. Store the solution as aliquots at −20°C.•3% (w/v) Paraformaldehyde solution (PFA) .Note 1. The PAF solution is prepared fresh prior to use. To prepare 100 mL solution, add 3 g of PFA powder in 80 mL water. To dissolve the PFA, add 100 μl of 1N NaOH and heat the solution in a 60°C water bath. Vortex to mix. Cool the PAF solution, add 10 mL of 10X PBS and 100 μl of 1N HCl solution to adjust the pH at 7.4. Add water to make the final volume to 100 mL and filter it to avoid particles. Keep the solution at RT until use.Caution: PFA is toxic and corrosive. Avoid any direct contact and wear appropriate personal protective equipment.•2 , and 1 mM EGTA  . Prepare the buffer in distilled water. The C buffer can be stored at 4°C for several months.C (CSK) buffer. It contains 100 mM NaCl , 300 mM sucrose , 10 mM PIPES pH 6.8 , 3 mM MgCl•Caution: Methanol is toxic if inhaled and in contact with skin. Avoid any direct contact and wear appropriate personal protective equipment.100% Methanol . •Caution: TCA is corrosive. Avoid any direct contact and wear appropriate personal protective equipment.100% Trichloroacetic Acid (TCA) . Store at 4°C. •100% Ethanol.•Blocking buffer: PBS containing 5% (w/v) albumin from bovine serum  and 0.1% Triton X-100.•Blocking buffer for PAR detection: PBS containing 5% (w/v) milk powder and 0.1% Tween 20 .•12 N HCl .•Wash buffer: 0.1% Tween 20 in PBS.•Caution: DAPI is mutagen. Avoid any direct contact and wear appropriate personal protective equipment.DAPI  stock solution (5 mg/mL) in distillated water. For long-term storage, the solution can be aliquoted and stored at −20°C. The solution can be keep at 4°C, protected from light, for short term storage. •1X Phosphate-buffer saline (PBS), pH 7.4.•Mounting solution .•Primary antibodies: mouse anti-CPD , mouse anti thymine dimers (T-T) , goat anti-DDB2 , and rabbit anti-PAR .•Secondary antibody conjugated to fluorescent dye: Alexa Fluor 488 and 594 goat anti-rabbit and anti-mouse IgG  and Alexa Fluor 488 donkey anti-goat IgG  are used at 1/500 dilution.•3% (w/v) Paraformaldehyde solution (PFA).•C (CSK) buffer.•2, 1 mM EGTA, 0.5% Triton X-100 . C+T buffer can be stored at 4°C for several months.C+T buffer (CSK buffer + 0.5% Triton): 100 mM NaCl, 300 mM sucrose, 10 mM PIPES pH6.8, 3 mM MgCl•C+T+S buffer : 420 mM NaCl, 300 mM sucrose, 10 mM PIPES pH 6.8, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100. C+T+S buffer can be stored at 4°C for several months.•100% Ethanol.•Blocking buffer: PBS containing 5% (w/v) BSA and 0.1% Triton X-100.•12 N HCl .•Wash buffer: PBS-0.1% Tween 20.•5 mg/mL DAPI stock solution.•1X PBS, pH 7.4.•Mounting solution.•Primary antibodies: mouse anti-CPD (1/1000), mouse anti thymine dimers (T-T) (1/2000), goat anti-DDB2 (1/500), rabbit anti-XPC , mouse anti-PARP1 , rabbit anti-XPA .•Secondary antibody conjugated to fluorescent dye: Alexa Fluor 488 and 594 goat anti-rabbit and anti-mouse IgG and Alexa Fluor 488 donkey anti-goat IgG (1/500).Note 2. Prepare the buffers in advance without protease or phosphatase inhibitors and PMSF and store it at 4°C and add the inhibitors on the day of the experiment. Alternatively, prepare the complete buffer and freeze it at −20°C.Note 3. Prepare in advance the protease and phosphatase inhibitors and store in aliquots at −20°C. Thaw them on the day of experiment.•1X PBS, pH 7.4. Store at 4°C.•100 mM phenylmethylsulfonyl fluoride (PMSF) : 17.42 mg/mL in isopropanol.•100 mM β-glycerophosphate disodium , Ser/Thr phosphatases inhibitor: 21.604 mg/mL (solubility limit 50 mg/mL) in distillated water.•1 M sodium fluoride , Ser/Thr, and acidic phosphatases inhibitor: 41.99 mg/mL in distillated water.•100 mM sodium orthovanadate , Tyr and alkaline phosphatases inhibitor: 18.391 mg/mL in distillated water. Set pH to 9.0 with 1N HCl and boil until colorless. Cool to room temperature. Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling. Bring up to the initial volume with distillated water.•10X protease inhibitors : dissolve one tablet in 1 mL distillated water.•2, 0.34 M sucrose, 10% glycerol , 0.1 % Triton X-100, 1 mM PMSF, 1X protease inhibitor, 10 mM β-glycerophosphate, 10 mM sodium fluoride, 1 mM sodium orthovanadate in distillated water.Buffer A: 10 mM Hepes, pH 7.8 , 10 mM KCl, 1.5 mM MgCl•Buffer B: 50 mM Tris pH 7.8 , 420 mM NaCl, 1 mM EDTA  , 0.5 % IGEPAL , 0.34 M sucrose, 10 % glycerol, 1 mM PMSF, 1X protease inhibitor, 10 mM β-glycerophosphate, 10 mM sodium fluoride, 1 mM sodium orthovanadate in distilled water.•2, 1 mM CaCl2 , 0.3 M sucrose, 0.1% Triton X-100, 1X protease inhibitor, 1 mM PMSF, 10 mM β-glycerophosphate, 10 mM sodium fluoride, 1 mM sodium orthovanadate in distilled water.Chromatin extraction buffer: 20 mM Tris-HCl pH 7.5, 100 mM KCl, 2 mM MgCl•Micrococcal nuclease solution > 100U/μl .•Benzonase solution 379U/μl .•10 mM MG132  stock solution. Dissolve at 4.76 mg/mL in DMSO. Solution can be aliquoted and store at −20°C.•10% TCA solution.•Solubilisation solution: 0.25N NaOH , 0.025% Triton in water.•22% Bradford assay dye solution  in distilled water. Store at 4°C.•Protein (BSA) standard solution (2 mg/mL) .•Antibodies: goat anti-DDB2 (1/1000), rabbit anti-XPC (1/1000), rabbit anti-XPA (used for IP at 1/100 dilution), mouse anti-PARP1 , rabbit anti-PARP1 (1/5000), rabbit anti-beclin , and rabbit anti-histone H3 .•Sterile 35- and 100-mm culture dishes (or other size) .•2 and 37°C).Cell culture incubator .•Microscope cover glass .•Isopore membrane filters .•Forceps.•Ultraviolet hand lamp EF-140 with UVC lamp (BLE-2537S) .•UVX digital radiometer .•Parafilm .•Inverted microscope  with Axiocam MRm camera.•Microscope slides .•1.5- and 15-mL tubes.•Pipettes, tips, and scrapers.•Refrigerated centrifuges.•Sonic Dismembrator Model 500 (Thermo Fisher Scientific).Protocols described here have been widely used in NER field for the last 20 years . They haA.1.1 Sterilize the glass coverslips. With sterile forceps, dip the glass coverslips in 100% ethanol. Air dry by place them in an angle into a 15 cm plate. Once they are dry, they are placed in 35 mm sterile dishes.Note 4. Prepare one coverslip for each experimental time point and controls. To determine the number of samples, one needs to understand that the normal NER kinetics at damage site (15 s to 3 h) is affected by gene manipulation. For example, the XPC half-life at damage site is 1 h, however, in XPA deficient cells, the level of XPC was stable from 30 min to the end of protocol (3.3 h) . Hence, Note 5. The buffers volumes are for one 35 mm dish.Note 6. Certain controls need to be run with each experiment, so plan to include these conditions when planning to seed the cells: IgG in place of primary antibody to check the specificity of the primary antibody, secondary antibody alone without primary antibody to verify lack of non-specific binding and no antibodies to verify autofluorescence.6) cells in total of 1.5 × 13 ml (19.5 ml) medium and we add 1.5 ml of this slowly on the glass coverslips. Before each pipetting, mix the medium well. Cells should be at 70-80% confluency at the time of treatment, however this can vary depending on the experimental setting.A.1.2 In order to prepare cells on coverslips, calculate the number of coverslips needed for the experiment and the total number of cells required. Scale up the cells to have 30-40% more cells than that required on the day of seeding the coverslips. Trypsinize the cells and take a cell count to ensure that there are enough cells to carry out the experiment and that the number of cells seeded are same in each experiment. In our experiments, we add 300,000 cells per dish in 1.5 ml medium. If we need to seed 10 dishes, we make a master mix for about 13 dishes, by suspending 300,000 × 13 .A.1.3 Grow the cells on the coverslip for 24 h in appropriate condition (5% COA.2.1 Pre-warm the UVC lamp for at least 10 min to avoid dose variability between the samples.2) × Time of exposure (sec). Place the dish within this region to accurately irradiate the cells.A.2.2 Measure the lamp flux using the UV meter across the surface of the irradiation. Determine the surface area required to irradiate based on the size of the dish used for the experiment. Measure the doses in five spatial points (the four corners and the middle of the irradiation surface) of this region and calculate the average to determinate the time needed to obtain certain dose of UVC, knowing that Dose = Intensity of the lamp  for global irradiation. Doses are applied by increasing the time of exposure.A.2.4 Irradiate the cells with a dose of 100 J/mA.2.5 Add media back to the dish and incubate for an appropriate time depending on the experiment setup.Note 9. Do not allow the cells to dry out during any steps, since drying will increase the background fluorescence. When a buffer is aspirated, the new buffer should be added quickly.Note 10. To stain the polymers of ADP-ribose, follow the steps A.3.6 to A.3.9.A.3.1 Aspirate the media and wash 2 times with 1.5 mL of buffer C.A.3.2 Fix the cells in 1.5 mL of 3% paraformaldehyde for 10 min at RT.A.3.3 Wash 3 times with 2 mL 1X PBS.A.3.4 Permeabilize in 1.5 mL of cold 100% methanol for 30 min at −20°C.A.3.5 Wash 2 times with 2 mL 1X PBS.A.3.6 Aspirate the media and wash with 1.5 mL of cold PBS.A.3.7 Fix the cells in 1.5 mL of 10% TCA in PBS for 15 min on ice.A.3.8 Aspirate the TCA and add 1.5 mL of cold 70% ethanol; incubate for 3 min at RT.A.3.9 Repeat the step A.3.8 using sequentially 90 and 100% cold ethanol.Safe stop! The coverslips can be stored at 4°C for 3-4 days in the blocking solution. In this case, seal the plate with parafilm strip around the lids to avoid evaporation and sample drying.A.4.1 Incubate the coverslips in 1.5 mL blocking buffer for 30 min at RT or overnight at 4°C. These steps will prevent non-specific antibody binding. Optional! Immunofluorescent labeling of CPD or 6-4PP in UV-damaged DNA requires denaturing of cellular DNA. To do this, after blocking, wash the coverslips 5 times with PBS and incubate them for 5 min at RT in 1.5 mL of 2 N HCl.A.4.2 Note 11. During this incubation time, prepare a humidified chamber by placing a moist paper towels at the bottom of a box or dish. Cover the paper with parafilm, so that the coverslips are not in direct contact with the paper towels. Since the coverslips will be placed on it, information necessary to identify the coverslip can be written on the parafilm using a fine permanent marker .A.4.3 Wash 5 times with 2 mL 1X PBS.Note 12. Do not aspirate the last PBS wash. Removing coverslip from a bufferless dish results in their breaking due to surface tension. After removing the coverslips, do not discard the dishes. The coverslips are placed in their respective dishes for washings between the antibody incubations.invert the coverslip over the antibody, placing it “cell side facing down” over the drop. Cover the box with a lid or an aluminum foil to keep the moisture in. Incubate for 1 h at RT or overnight at 4°C.A.4.4 Dilute the first antibodies in blocking buffer (40 μl of diluted antibody per 25 mm coverslip). Place 40 μl drops of the diluted antibody near each identified spot on the prepared parafilm. Remove the coverslip from the dish using pointed forceps and blot the excess buffer by touching the edges of the coverslips on a paper towel. Gently A.4.5 Using the same forceps, gently remove the coverslip from the humid chamber and place it “cell side facing up” in its original dish containing 2 mL of wash buffer.Note 13. Be very careful to not mix up the side on which the cells are grown.A.4.6 Wash with 2 mL of wash buffer 3 × 5 min each.Note 14. Subsequent steps must be done in the dark to minimize the photobleaching of the fluorophore. Cover the dishes containing the coverslips and the humidified chamber with aluminum foil.A.4.7 Dilute the secondary antibody at 1/500 dilution in blocking buffer. Proceed as describe in Note 11, 12 and step A.4.4. Incubate the coverslips in a humidified chamber for 30 min at RT.A.4.8 Transfer the coverslips back to the dishes and wash with 2 mL of wash buffer 3 × 5 min each.A.4.9 Prepare 1.5 mL of DAPI solution (0.25 μg/mL) in 1X PBS per 35 mm dish. Add over the coverslip in the plates and incubate for 5-10 min at RT. DAPI helps in microscopic identification of the nuclei by staining the DNA.A.4.10 Wash 2 times with 2 mL 1X PBS to remove excess DAPI.A.4.11 Wash 2 times with 2 mL distillated water to remove salts.A.4.12 Add 2 mL of distillated water in the dish and remove the coverslips with forceps. Remove the excess water by dabbing the edges of the coverslips over a paper towel. Let the coverslips air dry into a box on a paper towel.A.4.13 Mount the coverslips on a microscopic slide by inverting them on a drop of mounting media. The 20 μl drop of mounting media is placed on a slide using a 200 μl tip with a cut end. Take care to not introduce air bubbles while placing the drop and while placing the coverslip over it. Allow the antifade to slowly spread and cover the entire coverslip with minimal leakage outside the border of the coverslip. Seal the coverslips edges with clear nail polish to prevent drying and movement of the coverslips during image acquisition.Safe stop! The slides can be viewed immediately or stored at −20°C, in dark, for months.Note 15. An important issue in interpreting the results of the recruitment of protein in the damaged region of the nucleus is the quantification of the signal. In the local irradiation technique, since most of the NER proteins would be present in the nucleus even before irradiation, the extent of enrichment of the protein at the site of DNA lesion after irradiation is derived as fold increase in signal intensity at the lesion site that is identified by CPD signal over the signal for the same protein in equivalent area of interest in the portion of the nucleus that is not irradiated. To analyze hundreds of images of this type and obtain a very robust statistical data in shorter time, it is advisable to develop macros either for the microscopy software or freely available Image J program.A.5.1 Take the images of a least 100 nuclei at 40x or 63x magnification. We use an inverted fluorescence microscope Axiovert 200 (Carl Zeiss) equipped with AxioCam MRm.Safe stop! Data processing can be conducted as per convenience of the user.A.5.2 Delineate manually all the CPD positive spots using AxioVision 4.7 or others type of image analysis software.A.5.3 To quantify the level of protein at lesion site, measure its fluorescence at the CPD spots.A.5.4 Delineate and measure the intensity of a similar area in the unirradiated zone of nucleus to obtain a background corrected signal for the protein of interest.A.5.5 Subject the data for the intensity of at least 100 spots from three independent experiments to statistical analysis to determinate the significance of difference.Note 16. Cells are seeded and irradiated as described in local irradiation protocol; follow the steps A.1 and A.2.Note 17. Do not allow the cells to dry out during any steps, since drying will increase the background fluorescence. When a buffer is aspirated, the new buffer should be added quickly.B.1.1 Aspirate the media and wash 2 times with 1.5 mL of buffer C.B.1.2 Permeabilize in 1.5 mL of buffer C+T for 8 min at RT. This step will remove soluble cytoplasmic and nucleoplasmic proteins.B.1.3 Wash 2 times with 2 mL 1X PBS.B.1.4 Fix the cells in 1.5 mL of 3% paraformaldehyde for 10 min at RT.B.1.5 Wash 3 times with 2 mL 1X PBS.optional and recommended to extract the abundant proteins bound to undamaged chromatin.B.1.6 Extract with C+T+S buffer for 20 min on ice to remove the protein bound to chromatin under unchallenged condition. This step is B.1.7 Wash 3 times with 2 mL 1X PBS.Note 18. Follow the step A.4 described in local irradiation protocol.Note 19. Follow the step A.5 described in local irradiation protocol.Note 20. Before starting the procedure, prepare all your solutions and keep them refrigerated. Switch on the refrigerated centrifuges. Perform all steps on ice to avoid proteins degradation. When scraping the cells from the plates, place them on ice . Identify and prepare all the tubes for the cell collection and place them on ice.Note 21. Although the volumes are small, use 15 mL tubes and a centrifuge with a swing out rotor to obtain good pellets.Note 22. Seed at least 10 cm dish per condition. Cell should be at 80% confluency the day of irradiation. The buffer volumes are for one 10 cm petri dish.2 UVC by following the steps described in A.2, except step A.2.3 (without polycarbonate filter).Note 23. Irradiate the cells globally with 10 J/mNote 24. If using proteasome inhibitors to study protein degradation due to ubiquitination, the proteasome inhibitor MG132 is added at 5-10 μM to the cells, 1 h before treatment.C.1.1 Aspirate media and wash the plate once with 3 mL cold 1X PBS. Scrape the cells in 1 mL cold PBS and transfer them to a 15 mL tube placed on ice. Add another 1 mL cold PBS to the dish, scrape and pool with the previously scraped cells.g for 10 min at 4°C.C.1.2 Centrifuge at 600C.1.3 Aspirate the PBS and keep the tube on ice.C.1.4 Add 190 μl of cold buffer A to the cell pellet. Mix with P200 pipette several times and leave the tube on ice for 7 min. Remove an aliquot as whole cell extract (WC) (about 30 μl to which add 30 μl of 2X loading buffer).g for 5 min at 4°C.C.1.5 Centrifuge the extract at 1000C.1.6 Transfer the supernatant using a P200 to labeled Eppendorf tube. This is the cytoplasmic extract. Take an aliquot for Western (30 μl).C.1.7 Wash the pellet once with 190 μl of buffer A; mix using P200.g for 5 min at 4°C.C.1.8 Centrifuge the extract at 1000C.1.9 Discard the washing .C.1.10 Add 190 μl of buffer B to each tube, mix with P200 pipette and transfer the cells to Eppendorf tubes. The extracts should be viscous due to nuclear breakage and release of DNA.g) at 4°C for 30 min.C.1.11 Incubate the tube on ice for 30 min and centrifuge at maximum speed . Keep the pellet to extract chromatin.Safe stop! You can either extract the chromatin on the same day or freeze the pellet and extract it later. Freeze all others fractions at -20°C.Note 25. The chromatin bound proteins are removed by digestion with nucleases in chromatin buffer. It is important to optimize the removal of proteins bound to chromatin in the cell extracts. This can be done by resolving the DNA released after nuclease digestion on agarose gel.Note 26. To prepare the chromatin-bound protein fraction, we use 100 U/mL MNase or 25 U/mL benzonase. Higher amount of MNase (4000 U/mL) or benzonase (50 U/mL) can be used to digest the DNA down to mononucleosomal level.C.2.1 Suspend the chromatin pellet in 50 μl of chromatin buffer.C.2.2 The tight chromatin pellet is opened by mild sonication at lowest setting (setting 11 in Sonic Dismembrator Model 500 from Thermo Fisher Scientific) for 10 s. For higher volumes, time might be more, up to 15-20 s. If the clumps of chromatin persist, put the tube on ice after the first sonication, and then sonicate again, for 10 s. Usually one sonication is enough to take care of the clumps.C.2.3 Add 100 U/mL MNase to each tube (0.25 μl of 20 U/μl stock). Mix by vortexing. Incubate the tubes at RT for 40 min. Vortex intermittently.C.2.4 Stop the reaction with 5 mM EDTA (1 μl of 500 mM stock solution) and 5 mM EGTA (5 μl of 100 mM). Spin at maximum speed for 10 min and collect the supernatant into a new tube. This is the chromatin extract.Safe stop! You can either freeze the fraction at −30°C or proceed to the Western blot analysis.Note 27. Always validate the protocol by verifying the purity of different subcellular compartments before analyzing the movement of repair proteins, since many of them are present in more than one cellular compartment at the time.Note 28. Cell fractions are run on Western blots either based on their cell numbers or protein content. Protein estimation is done by using Bradford assay in a 96 well plate.C.3.1 Whole cell extract in C.1.4: Sonicate the whole cell extract fraction at setting 45 for 20 seconds to reduce viscosity. This can be either frozen for later use or used to estimate proteins followed by Western blot analysis right away.C.3.2 For protein estimation in WC, precipitate the proteins by adding 1-2 μl of the above prepared extract to 100 μl of cold 10% TCA on ice for at least 30 min. Centrifuge at 16000 g for 5 min. Wash the obtained protein pellet with ethanol to remove traces of TCA and dissolve it in 50 μl solution of 0.25N NaOH-0.025% Triton X-100.C.3.3 To estimate the proteins in nucleoplasmic and chromatin extract obtained in C.1.12 and C.2.4 dilute the fractions 1: 20 and 1:10 in NaOH-Triton X-100 buffer, respectively. This will prevent the interference of salt and detergents on protein estimation and also bring the concentration of the sample in the linear range of standard curve for protein estimation (5-150 μg/mL).C.3.4 Quantify the protein concentration using a Bradford assay or similar assay.C.3.5 Separate 5-10 μg proteins of each cellular compartment on 10 and 12% SDS-PAGE, transfer it in wet condition (100V for 90 min or 35V for 16 h) in Tris-glycine transfer buffer without SDS to a nitrocellulose membrane  and probe with primary antibody against known cytoplasmic (beclin 1/1000), nuclear , and chromatin bound protein (H3 1/2000) markers . We obseThe three simple methods described above do not require specialized reagents and equipment and can be readily performed in most laboratories. These methods will permit analyses of the fate and functions of various endogenous or exogenous proteins during NER. These functions range from recruitment and persistence or departure from the lesion, their interactions with other partners and role of PTMs in these processes. If one uses exogenous tagged NER proteins, note that PTM may be affected by the presence of tag, as observed for DDB2 and XPC , which w2 UVC through an isopore polycarbonate filter with irregularly distributed 5 μm holes  to reveal the signal for lesion-recruited proteins , This meThe improved protocol not only permitted quantification of PARP1 enrichment at the UVC-lesion of irradiated cells but also identified its localization to DNA for housekeeping functions in the unirradiated control cells . We alsoThe basic and improved local UVC irradiation techniques allow visualization of recruitment of NER proteins to the lesion site. Here, we describe an alternative approach of the global UVC-irradiation followed by sub-cellular fractionation and immunoblotting to examine biochemical and mechanistic studies related to recruitment, tracking intracellular movement of NER proteins and their interaction with other partners in different cellular compartments. This technique is suitable for study of NER of DNA damage caused by global UVC irradiation, as described here, but also by chemical agents or drugs in cellular models. In this protocol, it is important to incorporate multiple validation steps during the experiments to minimize the variability in data and to generate the quantitative data from Western blot analysis. These validation steps include quantification of protein extracts, validation of antibody and internal controls, detection of the combined linear range for the target protein for the detection methods and internal loading control . We have2 UVC to obtain whole cell (WC), nucleoplasm (Np), and chromatin-bound (Ch) protein fractions. Each of these subcellular fractions were separated on SDS-PAGE and immunoblotted for DDB2  induced binding of DDB2 to DNA lesions results in its auto-ubiquitination and proteasomal degradation by 3-6 h, followed by a complete recovery in 24 h , the reduction in DDB2 levels from 0.2 to 4 h was visible in the chromatin-bound protein fractions but not in the whole cell extracts (2). We noted that addition of proteasome inhibitor made no difference to the levels of DDB2 in the whole cell extracts up to 4 h, but suppressed its departure from the lesion site in the chromatin-bound protein fraction (Different PTM regulate the arrival and/or departure of repair proteins during NER . Couplin in 24 h . HoweverU6 cells . Howeverextracts . To detefraction .in situ extraction and immunofluorescence labeling. The accumulation of XPA at the local irradiation site was significantly reduced in the presence of PARP inhibitor and abrogated when PARP1 was depleted by shRNA, demonstrating that the recruitment of XPA to DNA lesion in NER is dependent on PARP1 and its catalytic activity (XPA was the first NER protein shown to interact with PAR  and the The pulldown of a known NER protein followed by mass spectrometry identification of the interactome of this protein is the most exploited technique for identification of new players in NER. Many repair proteins form complexes with other proteins at the lesion site after induction of DNA damage , but somTo exemplify, we examined the mode of interaction of XPA with PARP1 in different sub-nuclear compartments of GM skin fibroblasts before and after DNA damage . The XPAThe repair of damaged DNA is crucial for maintaining genome integrity. A plethora of proteins are recruited to the lesion site in an orchestrated fashion to detect and remove the damage from chromatin. Although we have a good knowledge of minimal set of protein required for GGR, it is not yet clear how the NER recognition proteins can find the lesions so quickly in the compact structure of chromatin . MoreoveThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.MR and RS performed the experiments and analyzed the data. All authors conceived and designed the project, wrote and revised the manuscript, and read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a shared affiliation with the authors at the time of review.


We performed this clinical trial to evaluate the efficacy and safety of apatinib and oral etoposide in patients with HER2-negative locally advanced or metastatic breast cancer (MBC).2 on days 1 to 10 for 21 days. The primary end point was objective response rate (ORR). Secondary end points included progression-free survival (PFS), disease control rate (DCR), overall survival (OS), and safety.Patients with HER2-negative MBC previously treated with anthracycline and taxanes and failed ≥1 prior chemotherapy regimens were recruited. The starting dose of apatinib was 500 and 425 mg in patients with ECOG scores of 0–1 and 2, respectively. The etoposide capsules were given at 50 mg/mThirty-one eligible patients were enrolled. The median follow-up time was 11 months. The median PFS for all patients was 6.9 months [95% confidence interval (CI) 6.0–7.9], and 6.9 months (95% CI 5.3–8.6) and 6.6 months (95% CI 1.4–11.7) for patients with apatinib 425 and 500mg once daily, respectively. The ORR was 35.5% (11/31). The DCR was 87.1% (27/31). The median OS was 20.4 months (95% CI 11.4–29.3). The median PFS of patients who had hypertension and proteinuria was longer than that for those without hypertension and proteinuria. The most common grade 3/4 treatment-related AEs were hypertension , fatigue , thrombocytopenia .Apatinib combined with etoposide capsules is effective and tolerable in heavily pretreated, metastatic HER2-negative breast cancer patients. A lower apatinib dose provide equivalent efficacy and reduced toxicity.https://clinicaltrials.gov/, identifier NCT03535961. Breast cancer is the most common malignant tumor and the second leading cause of cancer-related death in women worldwide . The medTumor angiogenesis is closely related to tumor growth and metastasis, and antiangiogenic strategies are some of the most important strategies for metastatic breast cancer. Bevacizumab has shown some efficacy in the treatment of metastatic breast cancer, and chemotherapy as a first-line and second-line treatment for HER2-negative metastatic breast cancer has significantly extended median progression-free survival (PFS) , 6. PrecApatinib is an oral small molecule tyrosine kinase inhibitor (TKI) that selectively inhibits vascular endothelial growth factor receptor 2 (VEGFR2), which exhibits some efficacy in triple-negative and non-triple-negative metastastic breast cancer by monotherapy , 11. OraTherefore, this study aimed to explore the efficacy and safety of apatinib combined with etoposide capsules in pre-treated metastatic HER2-negative breast cancer patients from the NCT03535961 trial.Patients included in the trial were 18-to-75-year-old females with histologically or cytologically diagnosed HER2-negative locally advanced or metastatic breast cancer who received at least one regimen of chemotherapy after metastasis, including taxane and anthracycline, with an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. The patients had measurable lesions as defined by the Response Evaluation Criteria In Solid Tumors (RECIST) (v 1.1). Patients who had previously received small molecule anti-angiogenic TKIs and patients with uncontrolled hypertension were excluded from the trial.The study was approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences and performed in accordance with the Declaration of Helsinki. Every patient signed written informed consent.2 on days 1 to 10 for 21 days per cycle. The tumor response was evaluated every 6 weeks according to RECIST v1.1 until the disease progressed or intolerable adverse reactions occurred. Blood pressure was monitored twice a day for the first 3 weeks and at least once a day after blood pressure stabilized. Routine blood and urine tests were performed weekly, physical examinations were performed every 3 weeks, liver and kidney functions were monitored, and electrocardiograms and tumor marker tests were performed every 6 weeks.The starting dose of apatinib was 500 and 425 mg in patients with ECOG scores of 0–1 and 2, respectively, and apatinib was taken orally each day. The etoposide capsules were given at 50mg/mDosage adjustment was recommended if hematological toxicity above grade 3 or non-hematological toxicity above grade 2 (except for hair loss) occurred, including withdrawal and reduction.2 on d1–10 and 35 mg/m2 on day 1–7, respectively. When adverse reactions of grade 3 and above occurred, dose adjustments were made according to the protocol. When adverse reactions were of grade 2 or below, researchers were allowed to adjust the doses according to the specific conditions. Adverse events were graded using Common Terminology Criteria for Adverse Events (CTCAE) 4.0.The first reduced dose was 425 mg/day and the second reduced dose was 250 mg/day for patients with an apatinib starting dose of 500 mg. Patients with a starting dose of 425 mg were adjusted to 250 mg/day first and 250 mg every other day second. The first and second adjustments of etoposide dose were 35 mg/mThe primary endpoint was the objective response rate (ORR), and the secondary endpoints were the disease control rate (DCR), PFS, OS, safety. The PFS duration was defined as the interval between the initiation of treatment and the last follow-up in patients with disease progression (PD) or to death from any cause, whichever occurred first. The OS was defined as the interval between the initiation of treatment and death for any reasons.Simon’s two-stage design with a one-sided α = 0.05 and 80% test efficiency was used to determine the number of patients that needed to be enrolled . Previoup <0.1 in the Kaplan-Meier single factor analysis were included in the Cox regression model for analysis. SPSS 23.0 and GraphPad Prism 7.0 were used for data analysis.Patients receiving ≥1 cycle of apatinib were included for survival and safety analysis. PFS and OS were estimated based on a Kaplan-Meier curve. A log-rank test was used to compare the median progression free survival (PFS) in different subgroups. Factors with st, 2017 to May 1st, 2019. Two patients withdrew their informed consent. Because one patient had only bone metastases and no measurable lesions, 31 patients were included in the final survival and safety analyses  3.5–24.3]. All patients had breast invasive ductal carcinoma, with a median age of 47 years (34–65). Treatment was discontinued in twenty-five patients due to disease progression, and six patients continued to receive treatment until the cutoff day. According to the RECIST version 1.1, no patients achieved a complete response in this study, eleven patients (35.5%) achieved a partial response (PR), the ORR was 35.5% (11/31), 16 (51.6%) patients achieved stable disease (SD) Table 2,p=0.56), respectively, which has no statistical significance. The median PFS of hormone receptor-positive and hormone receptor-negative patients was 7.4 months (95% CI 6.0–8.8) and 3.1 months , respectively. There was no significant difference in median PFS between patients with and without visceral metastasis (p=0.82). There were eleven (35.5%), eleven (35.5%), and nine (29.0%) patients who had received 1, 2, and 3 chemotherapy regimens, respectively. Their median PFS values were 6.6 months (95% CI 3.4–9.7), 6.9 months (95% CI 0.4–13.4) and 7.0 months , respectively. The results of the subgroup analysis are shown in p <0.1 in the univariate analysis were included in the multivariate analysis, and the hazard ratio (HR) of hormone receptor status on median PFS was 2.4 months , which reach a marginal statistical significance. The results of the subgroup analysis are shown in Sixteen (51.6%) patients with an ECOG score of two received an apatinib dose of 425 mg, and fifteen (48.4%) patients with an ECOG score of 0–1 received an apatinib dose of 500 mg; the median PFS was 6.9 months (95% CI 5.3–8.6m) and 6.6 months (95% CI 1.4–11.7), .Most of the adverse events were mild to moderate Table 3 Twelve (38.7%) patients experienced apatinib dose reductions due to adverse events. Of these, five patients were in the apatinib 500 mg group , of which four patients experienced twice dose reductions, and seven patients were in the 425 mg group  and received one apatinib dose reduction. Eleven (35.5%) patients had their doses modified during or at the end of the first cycle, and five (16.1%) (four patients had a second dose reduction) had their doses modified at the end of the second cycle. Two patients underwent dose adjustment for etoposide capsules.versus 2.6 months (95% CI 2.2–3.0), HR 0.28 (95% CI 0.1–0.8), p=0.022)]; median PFS was also significantly longer in patients with proteinuria than those without proteinuria   This study firstly explored the application of apatinib combined with etoposide capsules in locally advanced and metastatic HER2-negative breast cancer. In this study, the ORR was 35.5%, the median PFS was 6.9 months, and the median OS was 20.4 months.The median PFS associated with apatinib monotherapy for metastatic non-triple-negative breast cancer and triple-negative breast cancer was 4.0 months (95% CI 2.8–5.2) and 3.3 months (95% CI 1.7–5.0), respectively , 11. In Antiangiogenic drugs combined with chemotherapy increased the PFS in locally advanced and metastatic HER2-negative breast cancer after second-line treatment. The results of this study are equivalent to or even better than those of similar studies. In the RIBBON-2 study, the median PFS was 7.2 months in patients with metastatic HER2-negative breast cancer using standard chemotherapy regimens combined with bevacizumab for the second-line treatment . Treatmevs. 6.9 months, p=0.56) in the 500 mg group and 425 mg group, but fewer adverse events were observed in the 425 mg group. Therefore, we recommend 425mg apatinib as the starting dose for combination therapy. In addition, there was no significant difference in the median PFS between patients treated in the second-line, third-line, or further in this study protocol (p=0.82), indicating that apatinib and etoposide capsules can be used in patients who progress after multiline treatment.In this study, different starting doses of apatinib were given on the basis of the patient’s ECOG score. There was no significant difference in the median PFS . A cohort study of patients with metastatic gastric cancer treated with apatinib showed that the median PFS was prolonged by 24.5 days (86.5 versus 62 days) in patients who developed adverse reactions such as hypertension, proteinuria, and hand-foot syndrome within 4 weeks of taking the drug, and the median OS was extended by 2.2 months (p=0.022), and the median PFS in patients with proteinuria was extended by 4.1 months (p=0.036), but there was no statistically significant difference in overall survival. The trends of adverse reactions and curative effects in this study are consistent with previous studies, and it is worth expanding the sample size for further research.It is worth noting that this study showed that the occurrence of hypertension and proteinuria may be a positive predictor for efficacy. Previous studies have shown that the occurrence of adverse events such as hypertension and proteinuria may be one of the predictive markers for the efficacy of antiangiogenic drugs. In a clinical study of sunitinib in advanced renal cell carcinoma , patient2 months . In our This study did have some limitations. First, the number of patients enrolled was small, and there was a lack of control cases. Second, there might be bias in the population because this is a single-arm, single-center clinical trial.In summary, this study demonstrates that apatinib combined with etoposide capsules has a good effect in the second-line treatment of HER2-negative locally advanced and metastatic breast cancer with tolerable adverse reactions, suggesting that the combination deserves further phase III clinical research.The raw data supporting the conclusions of this article will be available from the corresponding author by request.The studies involving human participants were reviewed and approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences. The patients/participants provided their written informed consent to participate in this study.PY proposed ideas and designed studies study concepts, and review manuscript. Conception and design were performed by BX, PY, JW, and FM. All authors have contribution in collection and assembly of data. Data analysis and interpretation were done by NH, AZ, YS, JY, and XW. NH was a major contributor in writing the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by National Natural Science Foundation of China , and National Key Research and Development Program of China [grant number 2018YFC0115204].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alepidea amatymbica is used as a herbal medicine for the treatment of various diseases. As a result of its high medicinal value, this plant is being overexploited by herbal traders with little attention being paid to its conservation, which could lead to its extinction. Cultivation of Alepidea amatymbica was conducted to determine the appropriate planting depth and rhizome fragment length for the growth of this plant. The experiment was laid out in a Complete Randomized Block Design (CRBD) with two factors in a 6 × 3 factorial design. There were six levels of fragment length  and three levels of burial depth . Emergence rate, number of leaves, leaf area, and plant height, number of florets, rhizome length gain, rhizome weight gain, shoot moisture, and rhizome moisture were measured as growth parameters. The best overall yield in terms of plant height, shoot emergence, rhizome weight gain, number of florets and number of leaves was observed in 7.5 cm planting depth at 6 cm rhizome length. Four- centimetre rhizome length had the highest leaf area of 111.9 ± 3.5 cm2, 101.3 ± 3.5 cm2, 105 ± 3.5 cm2 at 2.5, 5, 7.5 cm planting depth respectively. Shorter fragment lengths showed high potential for vegetative propagation in terms of rhizome length gain at all burial depths. These results suggest that A. amatymbica can regenerate from buried rhizomes and they may contribute to the establishment of a protocol for propagation that could help in conservation of this plant to avoid its extinction Alepidea amatymbica is one such plant species that is heavily harvested and threatened by trade [A. amatymbica is widely distributed in the grassland of the Eastern Cape, KwaZulu-Natal and Mpumalanga Provinces of South Africa and other Southern African Development Community (SADC) countries, such as Swaziland, Lesotho and Zimbabwe. The plant generally grows in riparian zones, along drainage lines, and forest margins at about 850–2500 mm above sea level [The intensive harvesting of wild medicinal plants has resulted in over-exploitation, which is a primary threat to biodiversity . Over-exby trade . A. amatAlepidea amatymbica has a long history of traditional use for the treatment of conditions such as asthma, cold, cough, influenza, sore throat, chest pains, headache, diarrhea, wounds, rheumatism and stomach ache [A. amatymbica is considered the tenth most recognised medicinal plant and is usually used as remedy for asthma and cough. The extracts of A. amatymbica have also been reported to be active against Human Immunodeficiency Virus (HIV) related diseases [A. amatymbica rhizome and root dry mass [ach ache ,7,8. In diseases . Importadry mass .A. amatymbica rhizome by traditional healers and herbalists has caused extensive harvesting, which has resulted in the scarcity of the plant [A. amatymbica was ranked as the second most frequently traded herbal plant and it is in demand in the KwaZulu-Natal province. Dold and Cocks [A. amatymbica is regarded as the most prevalent plant in Eastern Cape markets and estimated that more than 1200 tonnes is sold annually in the region. Due to its popularity in the markets, A. amatymbica harvesters travel to distant locations in the wild searching for it so that it can be available to consumers [Alepidea amatymbica is being imported from Mozambique, Botswana, Zimbabwe, and Lesotho to South Africa, where it is scarce [A. amatymbica. The population is estimated to have declined by at least 30% over the last three generations due to persistent harvesting pressure for the medicinal trade and some loss of suitable habitat for afforestation and cultivation [A. amatymbica could be very common. However, its distribution has reduced due to overharvesting impacts on the plant. Due to the threat to its survival, A. amatymbica is of conservation concern in South Africa. One of the potentially effective measures for stemming over-exploitation of this plant is to encourage its propagation and cultivation.The high demand for he plant ,12 Accorhe plant , A. amatnd Cocks  reportedonsumers . Alepides scarce . Accordis scarce , there htivation . O’Connotivation  also repA. amatymbica have morphophysiological dormancy, which makes the germination of seeds difficult unless physiological dormancy is broken [A. amatymbica after storage for three years. Although, seeds has been used for cultivation of A. amatymbica, seedling production using rhizome explants has never been done. Seedling production through micropropagation of rhizomes as explants and at appropriate planting depth has been reported by many researchers for Laccosperma secundiflorum [Miscathus giganteus [Tussilago farfara [The successful cultivation of medicinal plants is determined to a large extent by germination ability of the seeds . Howevers broken . Accordis broken , temperadiflorum , Miscathiganteus  and Tuss farfara  to improM. giganteus [A. amatymbica would reduce harvesting pressure from natural populations, and it could help in the conservation of the plant, especially in the context of continued and possibly increasing demand. This study was therefore conducted to determine the appropriate rhizome length and planting depth suitable for the cultivation of Alepidea amatymbica, to encourage conservation of the species.Planting depth of rhizomes is an important factor in crop propagation practices because it influences the time of shoot emergence, vigour, and establishment . Rhizomeiganteus ,26 also iganteus . Howeveriganteus . SuccessA. amatymbica were collected in May 2014 from Emnyameni Village  in the Keiskammahoek area of the Eastern Cape Province in South Africa. The area is classified as the Amathole Montana grassland [Mature plants of rassland  natural A. amatymbica. Each fragment was weighed and tagged with different coloured plastic tapes to indicate the length of the rhizome and the weight before planting. The rhizome fragments used in this experiment were then wrapped in moist sterile tissue paper, placed in plastic bags, and taken to the greenhouse.The foliage was cut off at the crown and rhizomes were washed under running tap water to remove adhering soil particles. Rhizomes were cut with a scalpel into six fragment lengths of 1, 2, 3, 4, 5 and 6 cm. A ruler was used to measure each fragment length  of A. amA six by three factorial, randomised complete block design replicated three times was used in this study. The variables were the length of the rhizome and the planting depth. Three plots of different sowing depth  and treatments  were randomly assigned within the treatments. A total of 18 rhizome length fragments  were planted at a depth of 2.5, 5 and 7.5 cm in each pot.The different fragment lengths of the rhizome  were planted individually in plastic bags of 14 cm in diameter. Growth medium was added to the bottom of the plastic bag, and the depth was measured from the top of the plastic bag, using a ruler to ensure a correct planting depth. The fragments were then placed at the appropriate depth of 2.5, 5 and 7.5 cm and then topped up with soil. Each fragment was horizontally placed in the centre of each hole. The green house was maintained at 18–26 °C and 60% relative humidity. All plants were irrigated daily with tap water.A. amatymbica emergence rate (%); DAP = days after planting.The emergence of leaves was recorded three times per week  for 12 weeks . A plantLeaf area was determined by calculatingconstant .n = 6) and its three replicates, and the mean number of leaves for each depth (n = 3) was determined.A meter ruler was used to measure the shortest distance between the upper boundary of the main photosynthetic tissue on the plant and soil level . Leaves The number of florets per plant was counted and averaged for each treatment.n = 6) in each planting depth (n = 3) at the start and end of the experiment was determined for both nonregenerative and live fragments.The initial and final weight of the rhizomes were used to determine whether or not the fragment was alive. If the fragment gained weight, then it was considered alive. If the plant did not have any shoot and the rhizome had either lost weight or had decomposed entirely, then it was considered dead. The average weight of three replicates per rhizome fragment MoisWhere applicable, data were subjected to the analysis of variance (ANOVA) using SAS  package. One way ANOVA in SAS was used to compare various growth parameters among treatments. A two-way ANOVA was also used to determine the interaction between planting depth and fragment length on various growth parameters. Mean separation test was conducted using Duncan’s multiple range tests.p < 0.05) in cumulative shoot emergence among different rhizome lengths at 2.5 cm depth. There was no significant difference (p > 0.05) among the rhizome lengths 2, 3 and 5 cm. Six-centimetre fragment length produced higher cumulative shoot emergence. A significant difference in cumulative shoot emergence was observed (p < 0.05) among different fragment lengths at 5 cm planting depth. There was no significant difference (p > 0.05) between the rhizome length 4 and 6 cm. Similarly, there was also no significant difference (p > 0.05) with rhizome length 3 and 5 cm. The least cumulative shoot emergence was observed at 1 cm rhizome length. There was no significant difference (p > 0.05) on cumulative shoot emergence among different rhizome length at 7.5 cm planting depth. There was however, no significant difference (p > 0.05) on cumulative shoot emergence between rhizome length 2 and 5 cm. The highest cumulative emergence was observed at 6 cm rhizome length.The effects of planting depth and fragment length on shoot emergence are shown in p < 0.05) in cumulative shoot emergence among different rhizome planting depth in all the rhizome fragment length. At 2.5 cm planting depth had significantly higher (p < 0.05) shoots emergence in all rhizome fragment lengths. There was no significant difference (p > 0.05) in cumulative shoots emergence between 5 and 7.5 cm planting depth at 5 cm rhizome length. The least cumulative shoot emergence was observed in 7.5 cm planting depth in all fragment lengths.There was a significant difference (p < 0.05) in the average number of leaves among different rhizome lengths at 2.5 cm planting depth. One centimetre rhizome produced the lowest number of leaves while the highest number of leaves were produced at 3 cm fragment length at a planting depth of 2.5 cm. A significant difference (p < 0.05) was observed in the mean number of leaves among different rhizome length at 5 cm planting depth. There was no significant difference (p > 0.05) between the rhizome lengths 1 and 2 cm. The highest number of leaves were observed at 3 cm rhizome length while the lowest was at 5 cm rhizome length. At 7.5 cm planting depth, a significant difference (p < 0.05) was observed in a mean number of leaves among different rhizome lengths. There was no significant difference (p > 0.05) between rhizome length 1 and 2 cm, which had the lowest number of leaves. The highest mean number of leaves was observed at 6 cm rhizome length.The effects of planting depth and fragment length on the number of leaves are presented in p < 0.05) in the mean number of leaves among different rhizome planting depth for all the rhizome fragment lengths. No significant difference (p > 0.05) was observed on the mean number of leaves between the planting depths of 5 and 7.5 cm in 2 cm rhizome lengths. At a rhizome length of 6 cm, the highest number of leaves was observed at the deepest (7.5 cm) planting depth. On the other hand, the lowest number of leaves were obtained from the medium planting depth of 5 cm with 5 cm rhizome length.There was a significant difference (p < 0.05) in average leaf area among different rhizome lengths at a planting depth of 2.5 cm. The rhizome lengths of 2 and 3 cm had lower average leaf area and were not significantly different (p > 0.05). Similarly, there was also no significant difference (p > 0.05) in leaf area between rhizome lengths 1 and 5 cm. The highest leaf area was observed at 4 cm rhizome length. There was a significant difference (p < 0.05) in average leaf area among different rhizome lengths at 5 cm depth. Lengths 2 and 3 cm showed no significant difference (p > 0.05), and the same trend was observed between length 4 and 5 cm, which had high average leaf area. The 2 cm rhizome length produced the lowest leaf area in the 5 cm depth. There was a significant difference (p < 0.05) in average leaf area among rhizome lengths at 7.5 cm depth, however, fragment length 1 and 6 cm were not significantly different (p > 0.05). Four-centimetre fragment length had significantly higher (p < 0.05) leaf area while 5 cm rhizome length had the lowest leaf area.The effect of planting depth and rhizome length on the leaf area are shown in p < 0.05) in leaf area among different rhizome planting depths for all the rhizome fragment lengths. Shorter fragments lengths (1–3 cm) produced a higher leaf area at deeper planting (7.5 cm). At fragment length 4 and 6 cm, the highest leaf area was observed at a shallow planting depth (2.5 cm).There was a significant difference (p < 0.05) in the height of the plants among different rhizome lengths at 2.5 cm depth. No significant different (p > 0.05) was observed on the mean plant height between rhizome lengths 2 and 5 cm. The highest mean height was observed at 3 cm fragment length. There was a significant difference (p < 0.05) on the mean plant height among different rhizome lengths at 5 cm planting depth. There was no significant difference (p < 0.05) in plant height between the rhizome lengths 3 and 4 cm, which had a higher mean value of plant height than another fragment length. Similarly, there was also no significant difference (p > 0.05) in the mean plant height between 2 and 6 cm fragment lengths. The lowest mean plant height was observed in the 5 cm fragment length. There was a significant difference (p < 0.05) on the mean height among different rhizome length at 7.5 cm planting depth, and this difference was observed among the rhizome lengths 1, 2, 4 and 5 cm, which had lowest plant height values. A significantly higher (p < 0.05) mean height was observed at 6 cm fragment length.The effects of rhizome fragment length and burial depth on plant height is shown in p < 0.05) was observed in plant height among different rhizome planting depths for all the rhizome fragment lengths. Longer fragment length (6 cm) produced taller plants at deeper planting (7.5 cm). There was no significant difference (p > 0.05) in the plant height between 5 and 7.5 cm planting depth at 2 cm fragment length. The lowest plant height was produced at a medium planting depth (5 cm) at 5 cm rhizome length.A significant difference (A. amatymbica plants started flowering at 21 weeks after planting. The number of florets produced from varying rhizome lengths and planting depths are shown in p < 0.05) in the number of florets among different rhizome lengths at 2.5 cm depth. No significant difference (p > 0.05) was observed on the number of florets between 4 and 5 cm rhizome lengths. The highest number of florets was observed at 3 cm rhizome length. At 5 cm planting depth, there was a significant difference (p < 0.05) in the number of florets among different rhizome lengths, however rhizome lengths 3 and 4 cm did not show any significant difference (p > 0.05). Similarly, no significant difference (p > 0.05) was observed between rhizome lengths 1 and 2 cm. The highest number of florets was observed in the 6 cm fragment length. There was a significant difference (p < 0.05) in the number of florets among different rhizome lengths at 7.5 cm planting depth. There was, however, no significant difference (p > 0.05) among fragment lengths 2, 3 and 4 cm. The highest number of florets was observed in the 6 cm rhizome length.The cultivated p < 0.05) in a number of florets among different rhizome planting depths for all the rhizome fragment lengths. No significant difference was observed between 5 cm and 7.5 cm, planting depths at 2 cm fragment length. The highest number of florets was observed at deepest planting depth (7.5 cm) with 6 cm fragment length.There was a significant difference (p < 0.05) in rhizome length gain among different rhizome lengths at 2.5 cm planting depth, however, there was no significant difference (p > 0.05) between rhizome length 4 and 5 cm. The same trend was observed between rhizome lengths 2 and 3 cm. Six-centimetre rhizome length produced the lowest fragment length gain. A significant difference (p < 0.05) was observed in rhizome length gain among different rhizome lengths planted at 5 cm depth. However, there was no significant difference (p > 0.05) in the rhizome length gain produced between rhizome lengths 1 and 2 cm, which were longer than others. Similarly, there was also no significant difference (p > 0.05) in the rhizome length gain between rhizome lengths 4 and 6 cm. The lowest rhizome length gain was produced in the 5 cm length. There was a significant difference (p < 0.05) in the rhizome length gain of the rhizomes lengths planted at 7.5 cm depth. There was, however, no significant difference (p > 0.05) in rhizome length gain between rhizome lengths of 5 and 6 cm, which had the lowest length gain than others. Similarly, there was no significant difference (p > 0.05) in the rhizome length gain between rhizome lengths 3 and 4 cm. The longest final rhizome length was obtained from 1 cm rhizome length at 7.5 cm depth.The effects of planting depths and fragment lengths on rhizome length gain are presented in p < 0.05) in rhizome length gain among different planting depths for all the rhizome fragment lengths. One centimetre fragment length had the longest rhizome length gain in all burial depths. The longest rhizome length gain was observed in the 2 cm rhizome length at medium depth (5 cm).There was a significant difference (p < 0.05) in the rhizome weight gain among different rhizome lengths at 2.5 cm planting depth. However, there was no significant difference (p > 0.05) between rhizome lengths 4 and 6 cm, which were recorded the highest weight values. Similarly there was also no significant difference (p > 0.05) in the rhizome weight gain between 1 and 5 cm rhizome fragment lengths. The lowest rhizome weight gain was observed in the 2 cm fragment length at 2.5 cm planting depth. There was a significant difference (p < 0.05) in the rhizome weight gain among different rhizome lengths at 5 cm planting depth. The highest rhizome weight gain was obtained from 6 cm rhizome lengths while the lowest was obtained from 4 cm rhizome length. There was a significant difference (p < 0.05) in the rhizome weight gain among different rhizome lengths at 7.5 cm planting depth. The highest rhizome weight gain was produced from 6 cm fragment length while 1cm fragment length produced the lowest rhizome weight gain.The effects of planting depths and fragment lengths on rhizome weight gain are presented in p < 0.05) in rhizome weight gain in all rhizome fragment lengths. Shorter fragment length (1–3 cm) produced the highest rhizome weight gain at 5 cm planting depth compared to 2.5 cm and 7.5 cm plamting depth. Suprisingly the highest rhizome weight gain was produced at 5 cm planting depth by 6 cm long rhizomes. The lowest rhizome weight gain was also produced at 5 cm planting depth with 4 cm rhizomes fragment length.At all burial depths, there was a significant difference (p < 0.05) in moisture content among different rhizome fragment lengths at 2.5 cm planting depth. There was however, no significant difference (p > 0.05) in moisture content between rhizome lengths 2 and 5 cm. A similar trend was observed between fragment lengths 3 and 4 cm, which had lower moisture content. There was also no significant difference (p > 0.05) in moisture content between 1 and 6 cm rhizome lengths, which had higher moisture content values than others. There was a significant difference (p < 0.05) in moisture content among different rhizome lengths at 5 cm planting depth. There was however, no significant difference (p > 0.05) among rhizome lengths 2, 4, 5 and 6 cm. The 3 cm fragment length had the highest moisture content. There was a significant difference (p < 0.05) in moisture content among different rhizome lengths at 7.5 cm planting depth. Non-significant differences (p > 0.05) were, however, observed in the rhizome moisture content among rhizome lengths 1, 2, 3, and 4 cm, which recorded lower moisture content values. There was no significant difference (p > 0.05) in moisture between rhizome lengths 5 and 6 cm. Higher moisture content.was observed in 5cm fragment length.The harvest rhizome moisture content for different rhizome lengths and planting depths are presented in p > 0.05) in the rhizome moisture content of different rhizome fragment lengths. There was no significant difference (p > 0.05) in the rhizome moisture content in all burial depth between rhizome lengths 2 and 6 cm. One-centimetre rhizome length had the highest rhizome moisture content in the shallow burial depth (2.5 cm).At all burial depths, there was no significant difference (p < 0.05) in shoot moisture content among different rhizome lengths at 2.5 cm planting depth. Shoot moisture content among rhizome lengths 3, 4, and 5 cm was, however, not significantly different. The highest shoot moisture content was obtained from 6 cm rhizome length. A significant difference (p < 0.05) was observed in shoot moisture content among different rhizome lengths at 5 cm planting depth. There was however, no significant difference (p > 0.05) in shoot moisture content among rhizome lengths 2, 3 and 5 cm. Similarly, there was no significant difference (p > 0.05) between rhizome length 4 and 6 cm. The lowest moisture content was obtained from the 1 cm fragment length. Shoot moisture content significantly differed (p > 0.05) among different rhizome lengths at 7.5 cm planting depth. However, shoot moisture content among rhizome lengths 1, 2, 3, and 4 cm were not significantly different and recorded the lowest moisture contents. The highest moisture content was obtained from the 5 and 6 cm rhizome lengths.The rhizome shoot moisture content at different rhizome lengths, and planting depths are presented in At all burial depths, there was a significant difference among shoot moisture content of different rhizome fragment lengths. Shoot moisture content was the highest at 2.5 cm depth for all rhizome fragment lengths. The lowest shoot moisture was observed in 5 cm planting depth with 1 cm fragment length.A. amatymbica. At 2.5 cm depth, the emergence was found to be higher than at 5 and 7.5 cm depths regardless of rhizome fragment lengths. Previous studies on Alternanthera philxeroides (Mart) Standl. and Physalis viscosa L. also reported that deeper burial depth decreased the rate of emergence of shoots [Mentha arvensis (L) [Calystegia sepium (L.) R.Br. [Increase in planting depth significantly reduced the emergence rate of f shoots ,35. Thisf shoots  who showf shoots  showed tnsis (L)  and Caly.) R.Br. . One pos.) R.Br. ,40. The .) R.Br. . It has .) R.Br. .The current study showed that longer rhizome fragments increased shoot emergence rate. These results are consistent with a previous study . Other aThe present study showed that planting depth and rhizome fragment length influenced the growth and development of leaves, as it was observed that an increase in planting depth increased the number of leaves. Generally, 5 cm and 7.5 cm planting depth resulted in more leaves than 2.5 cm depth. These results suggest that there may be some stimulating effects of planting depth and fragment length on plant vigour. It has been reported that planting depth affects better utilisation of nutrients and greater ability to respond to favourable conditions which will result in more vigorous plant growth . SimilarA. amatymbica plants at 7.5 cm depth had the highest number of leaves, which compensated for the delayed emergence rate. This was also reported by Monaco et al. [A. amatymbica.In the present study, it was observed that 1, 2, 5, and 6 cm long rhizomes planted at 7.5 cm depth produced the tallest plants. This result could be explained by the fact that plants sowed at shallow depths often do not grow as vigorously as those sowed deeper . There io et al.  that as The plants originating from short fragments in all planting depths showed reduced growth performace, while long fragments resulted in higher plant productivity. This agrees with previous findings that suggested that non-structural carbohydrates and soluble proteins stored in the rhizome can be reutilised for the regeneration of plants that are subjected to disturbance ,52.Leaf area is an important variable for most ecophysiological studies in terrestrial ecosystems concerning light interception, evapotranspiration, and photosynthetic efficiency in plant growth . The groA. amatymbica. Plants buried at depths of 5 cm and 7.5 cm produced more flowers than those planted at a shallower depth (2.5 cm). Similar findings were reported by De Hertogh and Lenard [A. amatymbica formed flowers is an indication that rhizome cultivation will be a useful conservation method or approach for this plant.Flower initiation marks an important transition from vegetative development to reproductive development, which is the most crucial event in the life cycle of a plant. The flower produces the reproductive cells of plant and seeds for the next generation. The seeds enable the plant embryos to survive before seedlings establishment, thereby ensuring the initiation of the next generation of plants . Plant dd Lenard . The higd Lenard ,60. The A. amatymbica could reduce pressure on the wild population as a means of conserving this vulnerable medicinal plant and to address the demand for traditional medicine.Planting depth and fragment length had effects on the production of the rhizome biomass. The shorter fragments (1–3 cm) in this study had a higher yield on the rhizome weight at a planting depth of 5 cm. Similar findings were also obtained by Anbari et al.  who repoThe highest fresh rhizome weights observed in this study may be attributed to the development in plant height and a number of leaves which influence rhizome biomass. This study also validated the work of Asaeda and Nam . The difA. amatymbica cannot prevent its survival. Although sowing depth had an impact on the emergence of leaves A. amatymbica managed to recover from delay in the emergence, and responded positively to the planting stress. Shorter fragments at all burial depth of 5 cm produced the highest rhizome length gain. Medium planting depth (5 cm) is recommended for cultivation of A. amatymbica as it produces highest rhizome biomass required by traders, herbalists, and traditional healers. These results can play an important role in the establishment of propagation protocols of A. amatymbica. Vegetative propagation using rhizome could help to meet the current and future demands on A. amatymbica.The results of the study indicated that planting depths and fragment lengths of


Background and objectives: Obesity presents as a multifactorial, pandemic disease that arises as a consequence of unequal energy intake and energy consumption. Obesity adversely affects the quality of life, leading not only to disability, but also to various other disorders. Bariatric surgery is the most effective method for achieving significant and sustained weight loss in individuals with extreme obesity. The aim of this study was to examine how well surgically induced weight loss is maintained after five years of follow-up and its effects on cardiovascular risk factors and outcome. Materials and Methods: This is a retrospective cross-sectional study of 66 patients with morbid obesity, with body mass index (BMI) ≥ 40 kg/m2 or BMI ≥ 35 kg/m2 and obesity-related health conditions, aged 20 to 61 years, mostly women (77.3%) who underwent laparoscopic Roux-en-Y gastric bypass surgery. Results: Average follow-up was 6.42 years (95% CI 6.30–6.54 years) after surgery, with survival rate of 97% in operated individuals. There was a statistically significant reduction of weight and body mass index 6 months and 5 years after surgery in comparison to the initial values (p < 0.001). Of 62 patients who presented weight loss at the end of the follow-up period, 38 were able to maintain the amount of weight loss that was attained 6 months after surgery, while 24 patients regained weight compared to their postoperative weight at 6 months. Two patients reported no weight loss after treatment. Significant weight reduction was associated with better control of diabetes and increased self-reported physical activity at 6 months and 5 years after surgery, as well as with a reduction of the use of anti-diabetic and anti-hypertensive medications. Conclusions: Our research demonstrates a positive long-term impact of bariatric surgery on patients’ health conditions, significant and sustained weight loss, and decrease in BMI, which were associated with a reduction of co-morbidities and risk factors for cardiovascular diseases. Obesity is a common metabolic disorder, and its prevalence is increasing. It presents as a major public health problem in the 21st century /[BMI at surgery − 25 kg/m2]) * 100. All patients had their weight measured on an electronic Tanita scale type BC-401 , and their height was assessed with an altimeter. All patients were provided recommendations to engage in physical activity after surgery by having a minimum one-hour daily walk. Routine follow-up after bariatric surgery consisted of office visits twice a year, at which time the patients were assessed for blood pressure, electrocardiogram (ECG), and weight; basic laboratory blood tests were performed to determine fasting glycemia, lipide profile, and the levels of urea, creatinin, transaminases, electrolytes. Type 2 diabetes mellitus (T2DM) was diagnosed with two results of fasting glucose ≥ 7.0 mmol/L. We considered as index of diabetes resolution normal fasting blood glucose values of 4.0–6.1 mmol/L. According to the current recommendations, patients after bariatric surgery take supplements to ensure proper nutrition following their procedure including a complete multivitamin supplement, Vitamin B12, Calcium with Vitamin D, Iron and Vitamin C, Vitamin D. These vitamins help them to meet their nutritional needs. All supplements were prescribed on an individual basis in concordance with the clinical status of the patients.Local Ethical Committee approval was obtained. Sixty-six patients with morbid obesity with a BMI ≥ 40 kg/m (77.3%) ,13. Excl® (SPSS (Hong Kong) Ltd., Hong Kong). Categorical variables were presented as frequency of certain categories. The Chi-square test and McNemar test were used for analyzing the significance of the differences of categorical variables. Continuous variables are presented as median with interquartile range 25th–75th percentile or as mean with standard deviation according to data distribution and were compared using the nonparametric Wilcoxon Signed Ranks Test and Friedman test. Distribution normality was tested using the Kolmogorov–Smirnov test (the number of subjects was > 50). Overall survival estimates were calculated using the Kaplan–Meier method, and overall survival rates were presented as a mean ). The patients who remained alive were censored at the cut-off date. All analyses were estimated at p < 0.05 level of statistical significance.Statistical analysis was done using the statistical software package PASW Statistics 18The principles of ICH Good Clinical Practice were strictly followed, and approval from the Ethics Committee of the Clinical Center Serbia was obtained for the study protocol under the number 318/6 on 18 May 2017.Patients who underwent RYGB surgery were followed for at least five years, with some of them receiving follow-up for a longer duration. The mean duration of follow-up was 5.67 ± 0.63 years.p < 0.001, before vs. 5 years after surgery p < 0.001; BMI: before vs. 6 months after surgery: p < 0.001, before vs. 5 years after surgery p < 0.001). In addition to a statistically significant average body weight reduction after five years from surgical treatment, weight decreased an additional 4.5 kg between the 6 month and the 5 years’ time points. However, the change in average weight and BMI from six months to 5 years after surgery was not statistically significant .Weight and BMI before and after surgery are presented in In comparing the body weight of the 64 followed patients 5 years after surgery and before surgery, 62 patients (93.9%) experienced weight loss (31.50 (20.00–44.25) kg), while 2 patients presented weight gain (4 and 7 kg). BMI was also lower at 6 months after treatment and further decreased at the 5-year assessment .In assessing the degree of weight loss at the 5 years’ time point compared to the level of weight loss 6 months after surgery, of the 64 patients who were followed, 38 patients (57.6%) presented additional weight loss (12.00 (6.25–17.00) kg), while 24 patients (39.4%) had weight gain (9.00 (3.50–15.50) kg). p < 0.001). Of the initial cohort of 66 patients, only 2 patients presented no weight loss after treatment throughout the follow-up period.Most patients (89.1%) presented > 10 kg weight loss at the 5 years’ time point in relation to their baseline weight prior to surgery, while only 10.9% of patients showed weight reduction of less than 10 kg in the same period .Results were similar using %EWL as a recommended parameter of weight loss and bariatric surgery success. At 6 months after surgery, the %EWL was 55.38 (39.54–70.77), while at 5 years after surgery, the %EWL was 64.74 (33.10–83.15).p = 0.621). As for complications after surgery, none was reported.Risk factors and lifestyle changes before surgery and 5 years after surgery are shown in Our study has demonstrated that there is a favorable impact of bariatric surgery on certain cardiovascular outcomes and cardiovascular risk factors in patients with obesity. This study is unique in our country for its long-term follow-up, examining the effects of bariatric surgery on cardiopulmonary capacity and cardiovascular risk factors.In terms of survival after surgery, we reported a high rate of surviving patients, with 97% of operated patients being alive at least 5 years after surgery . There wIn a large Swedish prospective study of persons with obesity (SOS), during a follow-up period of 15 years, the overall mortality rate was 30.7% lower in the bariatric group compared with control subjects who did not undergo surgery. The most common causes of deaths reported were myocardial infarction and cancer ,15. In aA study by Flum and Dellinger examined short- and long-term outcome (up to 15 years) after bariatric surgery, showing a significant reduction in deaths from 16.3% to 11.8% in surgically treated patients compared to study controls who were not surgically treated . In a meA recent meta-analysis has shown that weight loss in patients with obesity with co-morbidities is associated with improvement in multiple domains of health, as well as with increased survival . AdditioThe lack of long-term follow-up has been reported as a limitation of such studies. Hence, we followed patients in our cohort for a minimum of 5 years.2 (median: 42.90 kg/m2), while in women it was 43.8 kg/m2 (median: 43.60 kg/m2). Following surgical treatment, at 6 months the BMI in men and women was reduced to 33.4 kg/m2 and 33.8 kg/m2 (median: 34.40 and 31.90 kg/m2), respectively [2 in their study, with BMI at 6 months postoperatively after laparoscopic RYGB of 33.1 kg/m2. In a larger retrospective Mayo Clinic study where 180 patients underwent RYGB surgery, similar results were obtained. After a 3-year follow up, BMI reduction was observed from an initial value of 49 kg/m2 to 33 kg/m2 [Weight loss is considered one of the main parameters in evaluating the success of bariatric surgery. In our study, the mean preoperative BMI in men was 43 kg/mectively . Smith eectively  reported33 kg/m2 .In terms of weight loss, we reported an average weight loss of 31.7 kg in men (median: 28.0 kg) and 29.3 kg in women (median: 27.0 kg) 6 months after surgery. Similar results were observed in the Schauer study, where the average weight loss obtained one year after RYGB was 29.4 kg, while for patients who had sleeve gastrectomy (SG) surgery, it was 25.1 kg, compared to only 5.4 kg in the untreated population . LikewisImportantly, bariatric surgery in our study group was found to lead to significant and sustained weight loss with beneficial effects on certain associated co-morbidities, as well to a reduction in certain risk factors for cardiovascular disease. After surgery, we found a significant decrease in the number of patients with Type 2 diabetes and insulin resistance. In the initial cohort with 36% of patients who had diabetes at baseline, T2DM persisted only in 14% of them 6 months after surgery ,13.Blanc et al. reported  completePories and coauthors  reportedIn a study by Buchvald et al. , hyperteOur patients significantly improved their level of physical activity after bariatric intervention. Many of them replaced a sedentary lifestyle with a more physically active one. From the initial 34.8% of the participants who were considered physically active before the intervention, physically active patients increased to almost 64% 6 months after surgery . PhysicaIn Olbers’ study  of 78 paIn a 5-year study of 66 patients who underwent bariatric surgery for obesity in Belgrade, Serbia, we demonstrated a positive long-term impact of bariatric surgery as well as notable and sustainable weight loss and BMI decrease which were associated with reduction of co-morbidities and risk factors for cardiovascular diseases. In our study population, there was a significant decrease in T2DM, affecting initially 36% of the subjects and, 5 years following gastric bypass surgery, 14% of them. In addition, a significant increase in physical activity level was also noted in our population, with 72% of patients considered as being physically active 5 years after surgery. Also, the number of patients using antidiabetic drugs was significantly reduced. Of our initial cohort of 66 patients, only 3% of the participants reported no weight loss after surgery. Considering the long-term quality of life of patients with obesity, as well as health costs ,35, thesThe number of patients included and followed up postoperatively was relatively small, but comparable to those of previously published studies ,37. Our