However, it has been suggestedthat capillary electrophoresis may be a better option for use withHDX. We performed in solution HDX on peptides and bovine hemoglobin(Hb) followed by quenching, pepsin digestion, and cold capillary electrophoreticseparation coupled with mass spectrometry (MS) detection for benchmarkinga laboratory-built HDX–MS platform. We found that capillarieswith a neutral coating to eliminate electroosmotic flow and adsorptiveprocesses provided fast separations with upper limit peak capacitiessurpassing 170. In contrast, uncoated capillaries achieved 30% higherdeuterium retention for an angiotensin II peptide standard owing tofaster separations but with only half the peak capacity of coatedcapillaries. Data obtained using two different separation conditionson peptic digests of Hb showed strong agreement of the relative deuteriumuptake between methods. Processed data for denatured versus nativeHb after deuterium labeling for the longest timepoint in this study also showed agreement with subunit interaction sites determinedby crystallographic methods. All proteomic data are available underDOI:  This combined reduction in temperatureand pH results in a decrease of the exchange rate by more than 5 ordersof magnitude, and subsequent manipulation of the samples is typicallyperformed under quench conditions to minimize the loss of the deuteriumlabel (out exchange) from the peptide amide backbone prior to themeasurement of deuterium incorporation.4 Sample handling steps following the quenching step have grown withthe complexity of the system being investigated and primarily includedigestion by acid-resistant proteases followed by desalting/concentrationwith a trap column but can also involve additional steps to facilitatethe protein digestion, such as protein denaturation, chemical or electrochemicalreduction of disulfide bonds, detergent depletion and lipid strippingfrom membrane proteins, and deglycosylation of glycosylated proteins.13 Today, HDX–MS workflows are dominated by variants of liquidchromatography (LC)–MS approaches based on a Peltier-cooledultra-performance liquid chromatography (UPLC) module.14 Costly commercial platforms for HDX–MSare abundant in structural biology research, where they offer automated,online sample handling and analysis by low-temperature UPLC separations.The main drawbacks of these systems are their cost, and due to thelow temperatures, high backpressures  becauseof increased mobile phase viscosity, and reduced separation efficiencyof LC (a result of resistance of mass transfer).16 This resistance to mass transfer not only degrades the separationefficiency of LC but also results in an increased risk of carryoverof injected material from prior injections as the interaction betweenpeptides and protease, trap, and analytical column stationary phasebecomes more difficult to disrupt. The carryover of material is detrimentalto HDX experiments as retained peptides lose deuterons as they areexposed to 100% H2O solvents, only to then elute duringsubsequent separations, confounding the apparent uptake kinetics.18 Despite all these drawbacks, LC remains the workhorse of HDX–MSworkflows although there have been a handful of attempts to employcapillary electrophoresis (CE) in HDX experiments.Hydrogen exchange studies ofproteins, have since its inception in the 1950s, have become a keytool for structural biologists.21 CE separates based on the differential mobility of analytes in anelectric field with a velocity dependent on their size and charge or deuterated sheath-flow additives and, as such,offer limited application to structural investigations.32 Microchip electrophoresis has been utilized for the separation ofseveral proteins following label, quench, and digestion steps, andthough the separation was performed at ambient temperature, the speedof the separations allowed for similar deuterium retention to coldLC separations.16 One advantage of CE thatcould make it suitable for HDX is that, in contrast to LC, the separationperformance is, theoretically, improved at lower temperatures, fortwo main reasons:CE is a well-establishedmethod for the separation of numerousclasses of molecules and is a valuable method for proteomics research.d charge1.22 In t33 reducing any longitudinaldiffusion of the analytes, as shown in the equation for electrophoreticmobility μ given by the Einstein relationq is the effectivecharge and r is the hydrodynamic radius of the analyte.One trade off of this advantage is the requirement for higher fieldstrengths to maintain the same CE current as achieved in separationsat room temperature or even warmer conditions.First, low temperatures result in increasedviscosity (η)of the BGE,N can be definedasV is the potentialdifference driving the separation, μ is the electrophoreticmobility of the analyte, and D is the diffusivityof the analyte. In turn, the diffusivity of an analyte in a liquidwith a low Reynolds number can be determined from the Stokes–EinsteinequationT is the temperatureand kB is Boltzmann’s constant.As shown by Ma and Horváth,33 combining Second, in CE,the number of theoretical plates N is directly proportionalto the voltage applied in CE and inversely proportional to the temperature T. It should be noted that a reduction of temperature fromambient to 0 °C results in merely ∼10% increase of N. While the theoretical improvement is small, CE appearsto suffer less from the low temperatures used to reduce out exchangein HDX experiments, in comparison to LC, where the backing pressureduring cold separation may approach the limits of what the systemfittings can sustain.Hence, the plate number In this seminal study, we present proofof principal of in-solutiondeuterium labeling of peptides and proteins followed by quenching,digestion, and separation in fused-silica capillaries at subzero-degreesCelsius temperatures using CE–MS. The platform is cost-efficientand easy to implement with any MS.2O , formic acid(99+% Pierce 28950), formic acid , aceticacid (Fisher A113-10x1AMP), dimethylformamide , ammonium acetate , D2O , urea ,pepsin from porcine gastric mucosa , and methanol were obtained from Fisher Scientific .Sodium hydroxide , high-performance liquid chromatography (HPLC) peptidestandard mix (Sigma H2016-1VL), hemoglobin (Hb) from bovine erythrocytes(EMD Millipore 374834-10 g), ammonium persulfate (APS) (≥98%Sigma 248614-5 g), and bradykinin (BK) acetate salt (≥98% SigmaB3259-5 mg) were obtained from Sigma-Aldrich .Ammonium bicarbonate , acetonitrile (ACN) , H34 withthe modifications provided below.CE separations were performed,and gas phase ions generated using a coaxial sheath flow CE–ESIinterface previously described,Figure S1). Separations were performedin the anode to cathode mode using 24 cm long fused silica separationcapillaries with 105 μm outer diameter (OD) and 40 μminner diameter (ID) .Hydrodynamic sample injections were performed using 5 psi N2 (g) backing for 3 s for both capillaries used in this study .The electrical circuit was connected to earth ground via the stainless-steelneedle of the sheath liquid syringe. For uncoated bare fused silica(BFS) separations, the separation capillary was conditioned betweeninjections for 6 min with 3/1 (v/v) H2O/ACN, 10 min with3/1 (v/v) 0.1 M NaOH/ACN, 10 min with 3/1 (v/v) H2O/ACN,and 10 min with BGE 3/1 (v/v) 1% formic acid (aq)/ACN delivered at20 psi of N2 (g) by connecting the separation capillaryinlet to a laboratory built acrylic holder for solution vials using1/4-28 male to Luer lock assemblies .For linear polyacrylamide (LPA)-coated capillaries, 10 min of BGEat 20 psi was used to condition the capillary between sample and standardinjections. The capillary inlet was rinsed with water after NaOH flushingand sample injection .A direct current high voltagepower supply  was manually operated to ramp the voltageover ∼7 s to 20 kV unless otherwise specified. Stable currentwas observed during the separations, and an Ohm’s plot showsthe separations occurred within the linear region of the voltage–currentcurve . The stage was controlled bymotion control software to position the ESI emitter at the mass spectrometer inlet. Stainless-steelhypodermic tubing  of length 50mm  was used for the ESI emitter assembly.A 2 kV electrospray voltage was applied directly to the stainless-steeltubing and controlled using MassLynx software . The sheath liquid flow rate was 500 nL/minusing a solution comprising 50% MeOH, 0.1% formic acid, and 50 nMleu-enk. The electrospray was visually monitored with a Dino-Litelong working distance microscope .A PEEK tee assembly  was housed in an aluminumblock for thermal regulation and attached to a piece of optical breadboardfastened to a linear ball screw gantry stage . The Peltier cooling module was controlled using a smallOEM precision Peltier temperature controller  coupled with TEC Service software.A Peltier cooling module  wasmounted to the surface of an in-house designed  and machined  aluminumblock holding the ESI emitter. A liquid CPU cooler  operated in extreme mode withCorsair iCUE software was fastened to a small aluminum heat spreaderon the hot side of the Peltier cooling module to facilitate heat exchange(2 (g) cone gas flow rate. The instrumentwas calibrated on the day of use using an appropriate dilution ofsodium iodide  in 50% MeOH and 0.1% formic acid delivered to the electrosprayemitter at 500 nL/min through the sheath liquid capillary. Unlabeledpeptides were analyzed in data independent acquisition (DIA) modeusing the UDMSE method with instrument settings previouslyreported for this instrument, unless otherwise stated.37Generated ions were analyzed on aSynapt G2–Si operated in positive ion mode with a mass range of 50–1200Da in resolution mode using a desolvation temperature of 22 °Cand 250 mL/min N2O, pH6.79. An aliquot (13.75 μL) was diluted 20-fold in either 50mM ammonium bicarbonate in H2O ,50 mM ammonium bicarbonate in D2O (pHread 6.27),or 6 M urea in D2O . The peptide standards were incubated overnight and quenchedby 20-fold dilution into ice cold 0.24% formic acid in 1/4 (v/v) ACN/H2O, which lowered the solution pHread to 2.85. Theintact Hb was exposed to the deuterated buffer for five timepoints. The temperature was maintained at22 °C in an Eppendorf Thermomixer Compact  for the three longest timepoints, and the deuteration reactionwas quenched by preparing a 20-fold dilution of an aliquot of thelabeled protein in ice cold 3.5 mg/mL pepsin with 1% formic acid inH2O, which lowered the solution pHread to 2.31.A pulse-label control was prepared by incubating an aliquot of thestock Hb standard for 50,000 s in the thermomixer and then performinga 5 s labeling step, as described above. The protein/pepsin mixturewas vortexed for ∼5 s and an aliquot placed in a PCR tube containingice cold ACN, such that the final concentration of ACN was 25% (v/v).The PCR tube was removed from the ice and placed in a laboratory-designed,in-house machined acrylic sample pod featuring an o ring for a gas-tightseal and connected to a N2 (g) in-house line . The assembled sample pod was connectedto the separation capillary inlet by passing the separation capillaryinlet  through a union assemblywith a tubing sleeve through a single ferrule  at the inlet side, anda sample plug was injected at 5 psi for 3 s. A 16 mm length of 2 mmOD, 1 mm ID PEEK tubing was secured with epoxy  to thejunction of the tubing sleeve and the F-120 ferrule to provide a gas-tightfitting to the acrylic sample and BGE pod using a 2 mm push-in fitting. When the pods are attachedto the device, 1–2 mm of the capillary inlet will be submergedin the solution in the pods, and upon introduction of N2 (g) into the pods, solution will be forced into the capillary. Afterinjection, the sample pod was removed, the capillary inlet rinsedwith water  and replaced withanother acrylic pod containing BGE in a stainless-steel vial connectedto both N2 (g) and the HVPS . A plug of BGE  was injected intothe capillary to push the sample plug into the thermostatic regionof the capillary. After the plug injection, the HVPS was ramped, andacquisition was started.For HDX–MS, stock solutions of either 2 mg/mL HPLC peptidestandard, 4.4 mM ATII with 4.4 mM BK or 120 mg/mL intact bovine Hbwere prepared in 50 mM ammonium bicarbonate in H37 with a modification of settingenzymes to “non-specific.” CE–MS data obtainedwith UDMSE were searched against a database of UNIPROTpeptide sequence entries for HBA_BOVIN, HBB_BOVIN, and PEPA_PIG. Completedata were further processed with the HX-DEAL module in Mass Spec Studiofor HDX-analysis.38Tables S1–S5 show summaries of the experiments accordingto the Gothenburg format.Peptic peptides were sequenced withPLGS 3.0.3 , as previouslydescribed,16 Here, we first present benchmarks of the CE platform with a focuson peak capacities for several separation conditions as well as theanalysis of peptide standards for HDX to demonstrate acceptable deuteriumback exchange. We then present the results of a bottom-up analysisof bovine Hb for five timepoints and two different separation conditions.After machining and assemblyof the cooled fused-silica capillaryplatform 1,we assnc) is defined as “themaximum number of peaks... [that can] be separated on a given column.”39 It is an entity that is dependent on the separationefficiency in both CE and LC. In CE, the capillaries have no packing,and thus, plate counts are not subject to Eddy diffusion as in LC.Hence, there is no mass transfer as the separation is based on theanalyte’s behavior in an electric field. This leaves the commonfactor of diffusivity as the single major source of band broadeningin CE separations. Recently, several nc calculations appropriate for use with microchip electrophoresisand CE have been derived.39Peak capacity is an important factorin maximizing the utility of generated data in HDX experiments. Peakcapacity  and last (ME) migrating standard peptides. When wesubsequently measured peptic digests of Hb under these two conditions,the nc averaged 120 on BFS and 175 onLPA capillaries. Thus, nc for the LPAcapillary exceeded that of the BFS capillary by almost 50%. The differencesbetween the BFS and LPA capillaries are not surprising as the LPAcoating has been used in electrophoresis since 1985 to eliminate soluteadsorption and electroosmotic flow.40 Inaddition to the increased peak capacity of the LPA capillaries, thesequence coverage of Hb is increased due to the disruption of adsorptionof hydrophilic peptides to the silanol groups of the capillary wall.The Kyte–Doolittle hydrophobicity indices of all identifiedpeptides present in the final Hb data set have been mapped in a hydrophobicityhistogram , and a clear trendof increased coverage of hydrophilic peptides is seen for the LPAcapillaries.41 This is well in line withprevious findings that more hydrophilic peptides are detected withCE versus reverse phase LC.42 We choseto use 40 μm ID capillaries as we have done so in our previouswork.43 The use of larger ID capillaries at subzerotemperatures has been reported to be feasible and with the resultthat more sample can be injected in a shorter plug length versus smallerlumen capillaries while attenuating the band broadening that typicallyaccompanies sample injection on larger lumen capillaries.33In ouranalysis of 16 wehave obtained values that are placed inside of this range ,we were able to compare the peak capacity of 0 °C CE to 0 °CLC and ambient microchip CE  and LC for HDX separations,is range 1. While ochip CE 1. While Dmax isthe deuterium content of the peptide standards, ND is the number of labile backbone amide hydrogens ofthe peptides , and Dfrac is thefraction of deuterium in the labeling buffer.The most frequently usedpeptide standards in HDX–MS, bradykinin (BK) and ATII, comigratedon a BFS capillary with the BGE used in this study; therefore, wealso performed analysis of a HPLC peptide standard mix. A crucialconsideration in HDX experiments is the back exchange (BE) of deuteronsunder quench conditions and during the separation step. BE was calculatedaccording to eq 45The peptide standardswere also assessed for BE, and the results for BFS and LPA capillariesat 0 °C and using 20 kV as separation potential are listed in 46 further work is needed to assessthe viability of such an approach with the short capillaries and lowtemperatures used in our HDX workflow. Improved sensitivity and thussequence coverage can also be obtained for CE-based proteomics platforms,as demonstrated by trace-sensitive analyses by the Nemes group, achievingamol to zmol sensitivity for peptides.48While ME is not traditionally used to assess BE inHDX–MSexperiments, it seemed appropriate to include it in the analysis becauseit was the slowest migrating peptide in the HPLC peptide standard,as well as the most hydrophobic peptide in the mixture. Three otherpeptides in the standard mixture were not assessed for BE: gly–tyr,and val–tyr–val as they were much shorter than any typicalpeptide used for structural analysis; leu-enk was not assessed asit was present in the sheath liquid for use as lock mass correction.While the chosen BGE composition was based on the minimum BE achievedfor peptide standards, this does not necessarily mean the BGEs werethe best choice for achieving maximum sequence coverage or peak capacity.During development, much time was spent on analyzing the DMF-containingBGE and BFS capillaries as these were able to separate and detect10 of 11 peptides from an HPLC peptide retention time marker standardmixture. Although the aprotic-modified BGEs showed promise for peptidemapping,18 During data analysis, we havenot observed any signs of carryover in the inspected mass spectrafollowing injections of standard peptides, intact Hb, or peptic digestsof Hb. Sample carryover was assessed by extracting ion electropherogramsfor the 18 most abundant identified peptic peptides of Hb from standardpeptide separations injected after peptic digests of Hb. The two mosthydrophobic and the two most hydrophilic Hb peptides according totheir Kyte–Doolittle hydrophobicity indices were also searchedfor as carryover in the following separations. The use of a peptidestandard containing two or five peptides in the assessment of carryoveris justified due to the fact that a failed injection can result innot seeing any signal from the analytes being injected, and therefore,the analysis of a successful blank injection could be indistinguishablefrom a failed blank injection. Observing peaks from the peptide standardsdemonstrates via peak shape and migration time, that the injectionwas successful and the capillary is performing normally. Since inCE we would only expect carryover from residual material on the outsideof the capillary, ensuring that the capillary inlet has been immersedinto the sample gives us the highest probability of observing carryover.An example of the lack of observable carryover in an HPLC peptidestandard separation following a standard capillary conditioning step(10 min 20 psi flushing with 10% HAc BGE) after the injection of thepeptic digests of a quenched 5 s labeling timepoint is shown in Table S7. For the assessment of carryover, theundeuterated peptide ion base peak m/z was extracted as an EIE ± 0.02 Da, as well as the m/z for every isotopologue expected between the basepeak of the deuterated peptide ion and the base peak of the undeuteratedpeptide ion. We searched data for carryover in such a manner on bothBFS and LPA capillaries operated at −5, 0, 2 °C, and ambienttemperature for separations using 5% HAc; 10% HAc; 20% DMF, 20% HAc;10% DMF, 10% ACN, 20% HAc; and 25% ACN, 1% FA. Carryover from HPLCpeptide standards (n = 14); a standard of ATII &BK (n = 7); intact Hb (n = 1); andpeptic digests of Hb (n = 9) was searched for inthe data file from the immediately following separation after onesequence of capillary conditioning. Additionally, during curationof the Hb data sets, more than 1400 spectra were inspected duringworkup in Mass Spec Studio. We found no legitimate instances of carryoverin these data.Assessingdeuterium BE required the manual inspection of summedmass spectra to obtain deuteron uptake values. Spectral signal frompeptides which have been retained in the system become problematicbecause as they sit in the protonated solvent of the separation modality,deuterons will exchange before the material ultimately reaches thedetector. There has been much effort to address this phenomenon inthe HDX community.28 Between sample injections, the replenishingof BGE by flushing multiple capillary volumes of fresh BGE for LPAcapillaries and the extended NaOH surface cleaning for BFS capillariesappears sufficient to prevent any carryover signal from being observed.This finding is not surprising based on the lowamount of sampleinjected into the capillary versus the amount of sample used for LC(tens of fmol vs tens of pmol), and it has been suggested previouslythat CE separations have the potential to overcome the issue of carryover.E analysis andused for the full HDX time course, whereas Figure S10).After selecting the best BGE compositionfor each of the capillary surfaces, deuterium labeling at five timepointswas carried out. The higher migration time RSDs observed for the BFScapillariesare likely due in part to the fact that collecting the triplicatetimepoint measurements on BFS capillaries spanned more than threeweeks and required multiple capillary replacements. The two most polarpeptides in the list, α 136–141 and β 35–40,were detected in the BFS separations, but by the 500 s labeling timepoint,the signals were unusable. Since the migration times and peak widthswere initially recorded during manual curation of these six peptidesfor the purpose of reporting D uptake 2, furthe16 However, here we areperforming a 400-fold dilution of the sample prior to injection whilestill injecting only ∼50 fmol of protein digest. This is ∼3orders of magnitude less material than typical recent HDX workflows(10–50 pmol).51Although BFS capillaries offeredfaster migration times, the analysistook longer than when using LPA capillaries because the BFS capillariesrequired more rinsing steps between sample injections. An additionalconsideration was residual NaOH left on the emitter after conditioningthe BFS capillaries. Adding a tip-rinsing station to the platform,such as that used by the Sciex Digital Picoview nanospray ion source,would improve the robustness of this workflow. In contrast to thework on BFS that spanned a long time, measurement of the completeHDX time course on LPA was performed within an intense 21 h time periodusing a single capillary. While the separation reproducibility wasnotably better on the LPA capillary, it should be emphasized thatthese measurements were performed with manual voltage ramping. Incorporationof automated voltage ramping along with instrument triggering willlikely result in better migration time reproducibility. Also, migrationtime alignment could be an option for these data sets to enable timesavings during data curation. While the separations in this studywere carried out on rather short capillaries, for complex mixturesand/or larger proteins where more resolving power is needed from theseparation, the benefits of a longer capillary for increasing sequencecoverage may be of added benefit. Furthermore, while it is uncommonthat a sample may be available at 120 mg/mL concentration, we chosethis concentration in line with the previous report for microchipelectrophoresis.52 We compared the relative uptake of deuteriumin Hb under native conditions with that after subjecting a sampleof Hb to denaturing conditions with 6 M urea . 53 Specifically, the residues R32–Y43 and L92–D127 onα-Hb and R29–E42 and D98–Q130 on β-Hb coincidewith where these two monomers bind to each other. Our data showeddecreased susceptibility for HDX for the native state of Hb comparedto the urea-exposed state in those regions (S11) We considerthis finding to validate our method as a promising tool for structuralproteomics, enabling characterization of molecular binding and conformationaldynamics, such as those encountered in protein–ligand interactions.Although Hb is a well-studiedprotein due to its importance forthe uptake of oxygen in the blood, we wanted to investigate how wellour HDX CE–MS platform performed in terms of mapping structurallyrelevant information. Additionally, it is suggested to benchmark newHDX platforms using standard proteins such as bovine Hb.We have provided proof of concept for low-temperatureCE–MSapplied to in-solution labeling HDX. Although BFS capillaries providefast peptide separations and minimal loss of deuterium from labeledpeptides, our findings show that LPA-coated capillaries are superiorfor HDX CE–MS. This rationale is based on the ability of LPA-coatedcapillaries to offer excellent peak capacity and largely improvedsequence coverage while requiring less instrument time and agreeingwith BFS experimental uptake rates. LPA capillaries are routinelyused for intact protein analysis and also lend support for the applicationof low-temperature CE–MS for intact proteins to be fragmentedby electron-capture dissociation or electron-transfer dissociation.Due to the nature of electrophoretic separations, we do not observecarryover. This happens due to the standard procedures for replenishingBGE by flushing the capillary with multiple volumes of BGE (or othersolvents) between sample injections and because of the simplicityof flow path components. The beneficial lack of carryover with oursystem stands in stark contrast to LC-based HDX platforms, which requirethorough assessment and time spent mitigating carryover. There remainmany avenues to pursue for further optimization of this platform,including, but not limited to, BGE optimization , a concentrating/desalting step, immobilized/embeddedprotease digestion, upgrading the Peltier element to allow even colderseparations, incorporating a sheathless electrospray interface, alternatecapillary coatings, and assessing longer or shorter capillaries. Additionalinvestigations into the tolerance of the separation for salts andsolutes common in protein chemistry will also be a focus of futureoptimization. Several compounds such as the reducing agent tris(2-carboxyethyl)phosphineand anionic lipids or detergents which prove detrimental to LC separationsshould migrate away from the mass spectrometer under the electrophoreticconditions employed here, and their impact on the CE separations willoccur in future optimizations.


Two formulations were developed in the form of an oral sachet containing probiotics, and their efficacy and safety were evaluated in adults with functional constipation.Lactobacillus acidophilus, Bifidobacterium bifidum and Lactobacillus rhamnosus (3 billion Colony Forming Units - CFU); and another with Lactobacillus acidophilus, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium longum, Bifidobacterium lactis, Lactobacillus casei, Bifidobacterium animallis (8 billion CFU). The participants were randomized in a 3-arm parallel study and one oral sachet was auto-administered once a day for 30 days.One formulation with P ≥ .05). Only one adverse event was observed in a patient of group 2, related to abdominal pain.Primary outcomes were improvement in increasing the frequency of weekly bowel movements and improvement in stool quality. Secondary outcomes were number of adverse events. In the first week one observed an increase in stool frequency and in the quality of stools, showing an improvement in constipation. No statistically significant differences were observed between the three treatment groups in relation to these outcomes (Clinicaltrials.gov number: NCT04437147.The two probiotic cocktails were effective in improving the symptoms of functional constipation, by increasing both the weekly frequency of evacuation and stool quality, and were deemed safe.  The medical history of patients with constipation should be analyzed together with parameters such as fecal consistency, defecating frequency, effort needed to defecate, feeling of incomplete evacuation, abdominal pain and discomfort, use of laxatives, surgical history, comorbidities, lifestyle and work activity. Functional constipation is based on symptoms of nonorganic origin and diagnosed by the diagnostic criteria of Rome IV. The Bristol Stool Scale can help patients assess and describe aspects of their stools, facilitating the recognition of constipation severity. Intestinal constipation negatively impacts the quality of life and can lead to significant costs in the search for treatments and purchase of laxatives.,6 The treatment of constipation is a challenge in the sense that osmotic, stimulating, irritating, and prokinetic laxatives are usually used. However, it appears that up to 47% of the patients are not completely satisfied due to inconsistent response to laxatives and concerns about their safety, adverse effects, taste, inconvenience, and cost.Of the gastrointestinal disorders, constipation is one of the most reported conditions in clinical practice. such as infectious diarrhea, diarrhea associated with antibiotics, diarrhea associated with Clostridium difficile, hepatic encephalopathy, ulcerative colitis, irritable bowel syndrome, functional gastrointestinal disorders, necrotizing enterocolitis, and functional constipation.–11Currently, it is known that there is an important interaction between microbes and intestinal physiology. Therefore, probiotics have been used to treat many intestinal disorders,Bifidobacterium and Lactobacillus in adults can result in intestinal constipation.–14 Consequently, the probiotics used in humans for the treatment of constipation are more often of the species Lactobacillus and Bifidobacterium. Lactobacillus and Bifidobacterium can shorten the migratory myoelectric complex period and accelerate small intestine transit, partly due to increased release of serotonin (5-HT) which has promotility effects.Although the human gut microbiome comprises >400 bacterial species, evidence has shown that a decrease in the population of Lactobacillus and Bifidobacterium in the treatment of intestinal constipation have observed promising results,–19 conclusions also supported by systematic reviews on the topic. However, there is no consensus on both the types of probiotic strains and their dosages for the treatment of constipation.,21 Strain selection is an important step in the production of a probiotic. Probiotics should have a beneficial effect on the host and remain viable throughout the product lifetime.Clinical trials evaluating different strains of Lactobacilli  and 4 strains of Bifidobacteria . The other probiotic cocktail integrated 2 strains of Lactobacilli (LA and LR) and 1 strain of Bifidobacteria (BB). Hence, this study aimed at confirming whether the dosage of probiotic strains increased the weekly frequency of bowel movements and the quality of stools, improving intestinal functional constipation in human subjects.The clinical trial reported herein evaluated the efficacy and safety of 2 different probiotic cocktails when compared to a conventional fiber treatment. One of the probiotic cocktails integrated 4 strains of ClinicalTrials.gov . This study followed the CONSORT 2010 checklist of information to include when reporting a randomized trial (available at http://www.consort-statement.org/media/default/downloads/CONSORT%202010%20Checklist.pdf), and other information is available in the published protocol of this protocol.The study entertained herein consisted in a single-center, randomized, double-blind and controlled clinical trial, registered in The parallel clinical trial worked with the hypothesis that the groups that received probiotics had a greater increase in weekly frequency of bowel movements and stool quality, being therefore more effective than the group that received the conventional fiber treatment. During a 4-week timeframe, 153 patients with functional constipation (51 patients per group) were treated as follows: the first group received a probiotic cocktail containing 3 billion colony forming units (CFU) of mixed strains of probiotic bacteria per sachet, the second group was treated with 8 billion CFU of mixed strains of probiotic bacteria per sachet, and the third group was treated with the conventional fiber treatment for constipation .The study was carried out single-center, at Dr Karin Häckel’s Gastroenterology Clinic, located in Sorocaba, State of São Paulo, Brazil. The recruitment of patients for this study was carried out through a collaborative effort between the University of Sorocaba and Dr Karin Häckel at the Clinic of Gastroenterology.in Campus, via mobile phone text messages, and social networks were also explored to promote the study. Recruitment of participants was carried out until November 30th, 2020.For the dissemination of the study, digital platforms were used, and letters were distributed via e-mail and After verbal and written clarification of the study, the participants that agreed to enter in the study signed the Informed Consent Form already approved by the Ethics Committee of the University of Sorocaba. All authors had access to the study data and reviewed and approved the final version of the manuscript.Eligible patients for the study were adults aged 20 to 80 year-old with clinical diagnosis of functional constipation according to the Rome IV Consensus. Participants entered the study only after granting written authorization.The Rome IV Consensus defines functional constipation as a dysfunction that manifests itself as difficult, infrequent, and incomplete bowel movements. Constipation must have started 6 months earlier and become more frequent in the past 3 months, including 2 or more of the following characteristics: involving <25% of bowel movements , feeling of incomplete evacuation, a sensation of anorectal obstruction, digital maneuvers to facilitate the removal of fecal content, <3 spontaneous evacuations/week and need for laxatives.The exclusion criteria were the presence of gastrointestinal diseases, use of antibiotics or dietary supplements containing probiotics or prebiotics in the last 15 days, and pregnancy.Participants were instructed to store the sachets at room temperature and to take a sachet before breakfast, by dissolving the contents of a sachet in 150 mL of water. To improve treatment adherence, phone calls and/or messages were sent to verify that participants were following the correct protocol and working as planned.During the clinical trial, the use of laxatives was prohibited. The sachets were auto-administered during a timeframe of 30 days for all participants. The study consisted of 3 parallel arms:Active comparator: 3 billion CFU of probiotic bacteria (3 × 109 CFU per sachet)–strains LA 02 ID 1688 (1 billion CFU), BB 01 ID 1722 (1 billion CFU), LR 04 ID 1132 (1 billion CFU), vitamin C (ascorbic acid) 45 mg, vitamin B1 (thiamin) 1.1 mg, vitamin B2 (riboflavin) 1.1 mg, vitamin D3  40.000.000 IU/g (34 µg), magnesium hydroxide 0.3 g, calcium carbonate 0.5 g, natural vanilla flavor powder 0.03 g, fructooligosaccharides (FOS) up to 3 g: one sachet/day for 30 days.Active comparator: 8 billion CFU of probiotic bacteria (8 × 109 CFU per sachet)–strains LPC 00 ID 1076 (1 billion CFU); Bifidobacterium longum (BL) 03 ID 1152 (1 billion CFU); Bifidobacterium lactis (BS) 01 ID 1195 (1 billion CFU); Lactobacillus casei (LC) 03 ID 1872 (1 billion CFU); Bifidobacterium animalis THT 010803 (1 billion CFU); LA 02 ID 1688 (1 billion CFU), BB 01 ID 1722 (1 billion CFU), LR 04 ID 1132 (1 billion CFU), vitamin C (ascorbic acid) 45 mg, vitamin B1 (thiamin) 1.1 mg, vitamin B2 (riboflavin) 1.1 mg, vitamin D3  40.000.000 IU/g (34 µg), magnesium hydroxide 0.3 g, calcium carbonate 0.5 g, natural vanilla flavor powder 0.03 g, FOS up to 3 g: one sachet/day for 30 days.Conventional fiber treatment: vitamin C (ascorbic acid) 45 mg, vitamin B1 (thiamin) 1.1 mg, vitamin B2 (riboflavin) 1.1 mg, vitamin D3  40.000.000 IU/g (34 µg), magnesium hydroxide 0.3 g, calcium carbonate 0.5 g, natural vanilla flavor powder 0.03 g, FOS up to 3 g: one sachet/day for 30 days.The follow-up of patients was of 1 week after the end of the use of probiotics. These stool types are categorized into slow transit (types 1 and 2), normal transit (types 3–5), and rapid transit (types 6 and 7).The changes in bowel frequency  were annotated in a Table that the patients filled in with information regarding the daily frequency of evacuation and type of stools . The stool form was collected using the Bristol stool form scale (BSFS), a simple tool to estimate intestinal transit time. The BSFS classifies stools into 7 categories, including type 1 ; type 2 (sausage-shaped but irregular); type 3 (like sausage but with cracks on the surface); type 4 ; type 5 (smooth bubbles with sharp edges); type 6 ; type 7 .The metric of analysis was the comparison between 0 and 30 days, considering that the number of effective bowel movements over 4 times a week is an effective value for the treatment and the quality of the stools from types 3 to 5.Adverse events are undesirable signs or symptoms that occur during the study and occurrences that may or may not be causally related to the treatment. All adverse events considered possible or likely related to the test product were registered in the patient form. The number of patients and of adverse events and serious adverse events were duly recorded.Serious adverse events are defined as fatal, life-threatening, disabling, resulting in hospitalization or prolonged stay, or resulting in malformation, whether related to the product under test or otherwise. According to previous studies, probiotics are safe and any serious adverse event that could be related to the products under test would be considered unexpected. Any unexpected serious adverse events were to be reported to the physician. Any serious adverse events that could be related to the product under test would immediately lead to the discontinuation of the product under test. The correlation was established using epidemiological statistics available on the OpenEpi website . As a 15% dropout rate is expected, 153 participants were included in the study, aiming to reach the completion of at least 132 participants. The participants that entered the study and the treatment schedule are displayed in Figure The sample size was calculated based on two relative means and their respective standard deviations related to the weekly increase in bowel movements and stool consistency considering the data reported by Del Piano et al 2010).0. The coRandomization was performed by Random Allocation Software. Participants were equally stratified into 3 groups, each with blocks of 9 participants.After confirming the eligibility and reading/signing the Informed Consent Form, the participants received an identical sealed opaque envelope with the randomized sequential number, by one of the researchers Fabiana Cristina Rosa Mitelmão (FCRM). The packaging differed only in the number of manufacturing batches. All groups received identical sachets , with no possibility of differentiating one batch from another.The physician Karin Häckel (KH) selected each participant according to the batch number corresponding to the randomization. Eligible participants were allocated . Study participants and the investigator KH that provided the treatment and collected the outcomes were blinded.To promote participant adherence to the study and complete follow-up, the text message or call was sent out after 15 days to find out how the treatment was and whether there was discontinuation or deviation from the intervention protocol. The following questions were asked: Why did you not continue the treatment? Have you had any adverse events, if any? Participants were instructed to fill in the correct form if any adverse events occurred and what kind of stools and daily rate of evacuation.Participants could also be removed from the study if the treatment was interrupted in any way for any reason, whether due to forgetfulness or undue intestinal disconfort. Unmasking and revealing the intervention during the study was allowed if there was a serious adverse event reported, and the physician would investigate if the product was actually the cause. Participants were allowed to withdraw from the study at any time at their own request or be withdrawn at any time at the investigator’s discretion for safety reasons.Data from the participant’s medical records were entered into a Microsoft Excel spreadsheet and the medical records were stored in a safe place for proof of the study. The data collection form was used to record patient data from all participants and completed by the researcher FCRM, who also registered the data in a Microsoft Excel spreadsheet. The results measured were verified, in duplicate, to ensure their quality.t test was used to to compare BSFS scores within groups at different times. The level of statistical significance was set at P < .05. Statistical analysis was performed using the software STATA v.14.2. (https://www.stata.com/stata14/) .The statistical analysis of variance test was used to compare BSFS scores between groups whereas the paired The project was filed under the number CEP-Single CAAE: 84003418.9.0000.5500 and was approved on 09/17/2018. Informed and signed consents were declared at the clinic participating in the research by the outcome evaluators KH. Personal information about the participants was collected and kept confidential before, during, and after the end of the clinical trial by only one of the researchers KH, which was delivered to the researcher responsible for it FCRM. The researchers stated that they had no financial interest whatsoever in this clinical study. All authors had access to the study data and reviewed and approved the final manuscript. Figure A total of 153 participants were selected and 132 were enrolled in the study after clarification of the clinical trial and signature of the informed consent form. Following randomization, 41 participants were treated with 3 billion CFU/sachet (Group 1), 49 participants were treated with 8 billion CFU/sachet (Group 2) and 39 participants were treated with the conventional fiber treatment (Group 3). A non-pharmacological treatment is the first-line management of constipation involving the use of fibers.P ≥ .05). The single adverse event that was reported in only one participant in Group 2, was related to abdominal pain.Table The study entertained herein demonstrated that treatment with a new probiotic cocktail of either 3 LAB strains  or 8 LAB strains , ingested once a day, for 30 days, increased the weekly frequency of evacuation and the quality of stools in patients, who reported improvement in functional intestinal constipation. In addition, the probiotic mixtures showed favorable safety profiles, as evidenced with the report of only one adverse event that was not considered a serious event.Therefore, combination of different LAB strains did not have an impact in either their efficacy and safety, compared to the conventional fiber treatment.Bifidobacteria and Lactobacilli, alone or in combination, had beneficial results in patients with functional intestinal constipation and are safe for consumption.–20,25These findings corroborate previous studies, which demonstrated a beneficial effect using strains of probiotics, alone or in combination, in patients with functional constipation or some other gastrointestinal disorder. In a trial study, Ibarra et al used Bifidobacteria for the treatment of intestinal constipation. The groups received capsules that contained the strains at 1 × 1010 CFU (high-dose group); capsules with the strains at 1 × 109 CFU (low-dose group); and placebo capsules. The only significant difference in adequate relief of constipation was observed between the high-dose and placebo groups. The results presented in the study reported herein indicated that even with a lower dose , there was an improvement in bowel function. The combination of strains can be an indication of greater effectiveness. Riezzo et al, in a clinical trial, employed ordinary artichokes or artichokes enriched with LP (daily dose of 2 × 1010 CFU) for 15 days with a daily dose of 2 × 1010 CFU). The trial showed a positive effect on symptoms in constipated patients after intake of probiotic-enriched artichokes. However, the number of patients (20) was small, as well as the timeframe of treatment (15 days). Lewis et al also used LP and Bifidobacterium longum to check their effectiveness ahead of irritable bowel syndrome (IBS). The authors concluded that L. paracasei and B. longum may reduce GI symptom severity and improve the psychological well-being of individuals, indicating that these strains can also be useful in the treatment of intestinal constipation. Preston et al reported a clinical trial for the relief of symptoms of irritable bowel syndrome, using three strains of Lactobacillus . For all efficacy endpoints, improvement of 30% or more vs. placebo was considered clinically significant. The trial focused on evaluating several GI disorders arising from irritable bowel syndrome and not only constipation, however, it can be stated that these strains were effective for various intestinal problems, including constipation. Martínez-Martínez et al reported a systematic review work involving prebiotics in the treatment of elderly people. Differently, the work presented herein did not make such restriction. Those authors found that the most used strains were Bifidobacteria with improvement in intestinal constipation in 10% to 40% of the cases. However, the population was restricted to elderly persons and, in addition, the original study designs displayed heterogeneity. The studies, in general, corroborate the proof of the effectiveness of the LAB strains used in the present study in alleviating intestinal disorders, such as constipation.According to the literature, The clinical trial reported herein had a strong adherence, with only 4 patients discontinuing the study due to losing follow-up (n = 3 participants), and 1 participant of the group that was administered with 8 billion CFU/g reporting abdominal pain. This participant reported, in the third week of use of the product, flatulence and abdominal colic. These adverse effects can occur with the use of probiotics, being reversible by simply interrupting its ingestion.A limitation of the study reported herein was that the results were based on self-reporting of symptoms by selected volunteers with functional constipation, as opposed to direct observation. In the research work entertained herein there was no use of a placebo control. This was yet another limitation of the study, as it may have masked the effects of probiotics in the evaluation of functional constipation.of Lactobacillus and Bifidobacterium were well tolerated and safe for consumption. In addition, the four-week consumption of the probiotic mixtures improved digestive symptoms, especially stool quality, in adults with constipation. According to the literature, multi-strain probiotics might be more effective because of potential synergy and additive effects among the individual isolates. However, despite the availability of multi-strain probiotics, not all have shown superior benefits. In this study, no difference was observed between Group 1 and Group 2, indicating that a high number of probiotic strains may not be necessary.Although there were no significant differences in the efficacy of the products, combination of strains  Probiotics can collaborate not only with constipation, but improve overall human health, in general, due to their immune-modulating potential, their resilience against pathogen invasion of the gastrointestinal tract, or their anti-inflammatory properties.Our findings will allow to use all the formulations as a treatment for functional intestinal constipation, since the symbiotic formulations which contained different concentrations of probiotic bacteria and the one which contained only prebiotic fibers did not produce statistical differences between them, that is, all were beneficial and had their goal achieved departing from the first week of treatment. The advantage of using only prebiotics is due to the low cost to the patient when compared to formulations integrating probiotic bacteria. Nonetheless, probiotics are live microorganisms with an expanded range of healthful activities. There is increasing evidence from the biological applications of probiotics for the maintenance and improvement of gut health, inhibition of pathogenic bacteria, and improvement of immune system and concomitant overall improvement of human health.In conclusion, both probiotic bacteria cocktails integrating either 3 LAB strains or 8 LAB strains improved the symptoms of intestinal functional constipation by increasing both the weekly frequency of evacuation and stool quality, as early as from the first week of treatment, with sustained improvements throughout the fourth week of treatment. Treatment with these probiotic cocktails was safe and well tolerated, with only one adverse event resulting in discontinuation.The results of this study are encouraging, but further studies are likely needed to support efficacy, safety, and durability of the effects.Conceptualization: Fabiana Cristina Rosa Mitelmão, Marta Maria Duarte Carvalho Vila.Formal analysis: Marcus Tolentino Silva.Investigation: Fabiana Cristina Rosa Mitelmão, Karin Häckel.Writing – original draft: Fabiana Cristina Rosa Mitelmão, Cristiane de Cássia Bergamaschi, Victor Manuel Balcão, Marta Maria Duarte Carvalho Vila.Writing – review & editing: Cristiane de Cássia Bergamaschi, Marli Gerenutti, Victor Manuel Balcão, Marta Maria Duarte Carvalho Vila.


Isoamylase (ISA) is a debranching enzyme found in many plants, which hydrolyzes (1-6)-α-D glucosidic linkages in starch, amylopectin, and β-dextrins, and is thought to be responsible for starch granule formation (ISA1 and ISA2) and degradation (ISA3). Lipid-modified PEI (lmPEI) was synthesized as a carrier for long double-stranded RNA , which targets the three isoamylase isoforms. The particles were applied to the plant via the foliar spray and were differentially effective in suppressing the expressions of ISA1 and ISA2 in the potato leaves, and ISA3 in the tubers. Plant growth was not significantly impaired, and starch levels in the tubers were not affected as well. Interestingly, the treated plants had significantly smaller starch granule sizes as well as increased sucrose content, which led to an early sprouting phenotype. We confirm the proposal of previous research that an increased number of small starch granules could be responsible for an accelerated turnover of glucan chains and, thus, the rapid synthesis of sucrose, and we propose a new relationship between ISA3 and the starch granule size. The implications of this study are in achieving a transgenic phenotype for endogenous plant genes using a systemic, novel delivery system, and foliar applications of dsRNA for agriculture. The potato is an extremely important staple crop for humans, is grown and consumed in vast quantities, and is the only staple tuber crop grown outside of the tropics . PotatoePotatoes are carbohydrate-rich tubers, which contain approximately 80% water and 20% dry matter, of which, 60–80% of the dry matter consists of starch . In shorArabidopsis [Arabidopsis ISA3 loss of function mutant exhibited a starch-excess phenotype, while the simultaneous suppression of ISA1, ISA2, and ISA3 by transgenic hairpin RNAi resulted in a starch-deficient phenotype, despite the suppression of ISA3 which, according to the current literature, is responsible for degrading starch [Starch granule initiation is believed to be due to isoamylase activity . There abidopsis . Currentbidopsis ,6,7. Stabidopsis ,6. Thesebidopsis ,6. The rg starch ,5,6,7.Ever since the development of the first genetically-modified (GM) crop in the 1990s, commercial GM traits have been produced in 15 crop plants, and in 2015, over 18 million farmers from 28 countries planted almost 440 million acres of GM crops [In improving the plant performance, not the insect or pathogen resistance, in order to deliver traits with sufficient impacts to be economically viable, development should take into account germplasm and environmental niches and, thus, requires compensatory decreases in other product development costs . The neeSIGS is the topical application of double-stranded RNA molecules (dsRNA), which has the advantage of cheaper manufacturing of source RNA and the ability to rapidly customize and synthesize various gene targets  . SIGS noHowever, there are many issues facing the commercialization of SIGS technology. One of which is that application of naked dsRNA is prone to RNA degradation and poor penetration, as well as shortened silencing duration and non-specificity ,10. SomeHere, we report on the simultaneous targeting of ISA1, ISA2, and ISA3 using a single spray with a lmPEI nanoparticle dsRNA carrier and SIGS approach. A transgenic phenotype similar to previous research with a transgenic chimeric construct  was achiLeaves treated with the gene-silencing spray targeting ISA1, ISA2, and ISA3 had variable reductions in gene expressions, and in the case of ISA3, no effect at all in the leaves. As seen in p ≤ 0.05), which was not suppressed at all in the leaves as shown in p = 0.19) in the tubers as well.Surprisingly, gene suppression in the tubers did not match the suppression activity of the spray in the leaves. As seen in p = 0.06 and 0.1, respectively) as seen in The suppression of ISA1 and ISA2 in the leaves and ISA3 in the tubers did not significantly affect the yield, although there was a trend in the treated plants toward fewer potatoes and a median potato weight almost twice that of the control , and a large shift in the distribution of particle sizes toward less than 400 pixels2 as determined by the ImageJ analysis. On average, 1000 particles per biological replicate were examined, for a total of about 29,000 particles analyzed for the entire experiment.Most surprisingly, the treated tuber group, which did not show a significant reduction in ISA1 expression, but did show a significant reduction in ISA3 expression  still exRepresentative micrographs  in the treated potatoes; the median number of sprouts treated was approximately four, while the control potatoes had a median of two, as seen in The number of sprouts was significantly higher (p ≤ 0.05), water content, and the average weight of the whole plant , the total starch content and number of potatoes , total protein, and total sugar , and total sugar and number of sprouts .In order to test the data robustness, linear regressions were performed for the recorded variables in this experiment in order to explore their relationships with one another, as seen in the Pearson correlation heatmap in This is the first occurrence, to our knowledge, where a gene in a tuberous crop was successfully suppressed through the exogenous application of dsRNA to the leaves, although the gene silencing observed was not as efficient as transgenic methods . Furtherp = 0.06) and larger median potato weight (p = 0.10).We observed a phenotype similar to that observed when ISA1, ISA2, and ISA3 were silenced with transgenic hairpin RNAi , althougIn the current literature, there are many examples of SIGS for use against viruses, fungi, and insects . HoweverThe continued suppression of ISA1 and subsequent increase of ISA2 expression observed in the leaves  also reaArabidopsis thaliana genes, where it was found that one targeted gene was successfully silenced via siRNAs derived from exogenous dsRNA, while another targeted gene’s siRNAs were degraded and the gene was not effectively silenced [The sequence choice likely also played a role in the efficacy of gene suppression observed . One stusilenced .Solanum tuberosum tuber, which to the best of our knowledge, is not present in the current literature. Additionally, there has not been a mutant analysis of ISA3 in potatoes, only a complete loss of the function mutant, isa3 in Arabidopsis thaliana, which observed a starch-excess phenotype in its leaves [Solanum tuberosum.Literature studies today attribute decreased starch levels, granule sizes, and increased sucrose to the silencing of ISA1 and ISA2 ,6,7. Thes leaves ,6. Givens leaves ,6,7, theIn this work, we tested the hypothesis that a lower content of starch, achieved through the suppression of the three isoamylase genes, would lead to a metabolic flux toward sucrose sinks, more aromatic amino acid production and a higher protein concentration in the potato tuber. The results show a good correlation between the total sugar content and total protein . InteresA full experimental outline summarizing the number of biological and technical replicates is included in p ≤ 0.05. Significance for the cumulative granule size distribution was determined by Kolmogorov–Smirnov tests for the equality of distributions and a significance level of p ≤ 0.05. For testing the differences in the means, a regression analysis was performed for univariate and robustness, as well as a multivariate regression, which controlled for the number of potatoes, median potato weight, and water content on a per-plant basis. We controlled for these variables because they are variables likely to influence the measured outcomes and at the same time could be affected by the treatment. For the same reasons, similarly sized and shaped potatoes were chosen for the analysis. Lastly, a correlation heatmap was created between the number of potatoes, median potato weight, average weight of the whole plant, water content, total starch, total protein, total sugar, median granule size, and the number of sprouts on a per-plant basis.All statistical analyses were performed using JASP  with oneSolanum tuberosum L. cv. ’Desiree’ were vegetatively propagated from sprouting tubers in a mixture of 50% universal potting soil  and 50% perlite #4  with day temperatures of 23 °C and nighttime temperatures of 20 °C in a greenhouse with natural lighting conditions from the months of February to May. Plants were individually grown in 4 L pots with 20–20–20 fertigation through drip irrigation three times per day. Irrigation was stopped at week 12 of growth until the leaves were desiccated. Tubers were harvested at week 15, and stored in darkness at room temperature. Leaves were sampled bi-weekly for RNA prior to each treatment application and flash-frozen in liquid nitrogen.Every two weeks, for a total of six sprays over a 15-week growth period, the treated plants were sprayed. In order to ensure consistency between the sprays, the dsRNA-lmPEI solutions were vigorously vortexed and the three individual solutions for each gene were mixed into the handheld sprayer . The plants were sprayed at the same time of day until seepage was reached on each individual plant. Additionally, RNA sampling was performed prior to the bi-weekly spray in order to judge the effect of the prior spray and also to ensure that there was not a localized high concentration of dsRNA on the flash-frozen leaves, which could interfere with the RT-qPCR analysis or prevent the slow-release of dsRNA into the plant after the spray.At harvest, tubers were characterized and weighed individually, then similarly-sized and shaped tubers were split into groups to monitor sprouting behavior and metabolite analysis. Tubers of sizes 20–60 g and similar shapes were taken for metabolite analysis, while larger tubers were separated for sprouting behaviors.For metabolite analysis, approximately 80 g of tubers per biological replicate were weighed, washed, and dried with paper towels. The tubers were then cut into slices, blended using an immersion blender , and split for the analysis of the starch granule size, protein, starch content, flash frozen for RNA extraction, and lyophilized for water content and sugar determination. All materials for RNA were stored at −80 °C until extraction.In order to target the three forms of the isoamylase gene ISA1, ISA2, and ISA3, 250 bp-long sequences were chosen (shown below) in order to trigger the dicer-like protein at different locations along the protein and generate multiple siRNAs as previously described . All thrISA1:(NCBI accession number NM_001288008).TTT TTT GAG ATT TTG CGG CCT CAT GAC CAA ATT CCG CCA TGA ATG TGA ATC ACT GGG ATT AGA TGG TTT CCC TAC AGC AGA AAG GCT GCA ATG GCA TGG TCA CAC TCC TAG AAC TCC AGA TTG GTC TGA AAC AAG TCG ATT CGT TGC ATT CAC ACT GGT CGA CAA AGT GAA GGG AGA ACT ATA TAT TGC CTT TAA CGC CAG CCA TTT GCC TGT AAC GAT TAC ACT TCC AGA TAG GCC TGG TTA TAG ATGGISA2: (NCBI accession number XM_006355814).CCC CTG CAT ATG ATG CTC GAA AAT CTT TGG GTT GGA ATA CTT TAA AAA CTG GTT TTG GGA CTC AGA TTG CCC AGT TTA TTT CAT TCT TGA GTA ATT TAA GAA TGA GAA GAA GTG ATC TTC TTC AAA AGA GAA CCT TCT TGA AGG AAG AAA ACA TCC AGT GGC ATG GGA GTG ACC AAT CTC CTC CGA AAT GGG ATG GCC CGT CTA GCA AAT TCT TGG CTA TGA CTT TGA AGG CCG ATG CTG AAG TCA GCCAISA3: (NCBI accession number NM_001288291).ATG ATG CAA ACG GTG AAG GTG GCA ATG ATG GAT GCA ATG ACA ACT TCA GTT GGA ATT GTG GAA TTG AAG GTG AAA CTT CAG ATG CAA ATA TTA ACG CAC TGC GTT CAC GGC AAA TGA AAA ATT TTC ATT TGG CAC TGA TGG TTT CTC AGG GAA CAC CAA TGA TGC TTA TGG GGG ATG AGT ATG GGC ATA CCC GCT ATG GAA ATA ATA ACA GTT ATG GAC ATG ATA CCG CCA TCA ACA ATT TCC AGT GGGGThe chosen dsRNA sequences were synthesized, ordered , and complexed with lmPEI, as previously described . In shorThe dsRNA-lmPEI particle stability was also previously tested  using geubi3, L22576.1) were determined by RT-qPCR with the primers described below [RT-qPCR was performed with 15 biological replicates for each control and treatment group, and at least 3 technical replicates unless otherwise stated. Expression levels for the three isoamylase genes and reference gene ubiquitin  according to the manufacturer’s instructions. Total RNA was extracted from potato tubers as described . After ew/v) was then added to the separated and cleaned starch granules. This solution was vortexed until all granules were visibly stained.Starch granules were analyzed in a similar manner as previously described . After bGranules were diluted and pipetted onto microscope slides and analyzed with light microscopy; 7 micrographs of each biological replicate were taken using an Olympus U-CMAD3 microscope , an Invenio 5DII microscope camera, and DeltaPix InSight software  at 4× optical zoom. The surface areas of individual granules were calculated with ImageJ  as previIn a 50 mL tube , 25 g of de-ionized water was added to 0.25 g of freshly blended potato in triplicate for each biological replicate. As described , the mixA calibration curve was created with pure potato starch from Merck  in concentrations ranging from 0.01 mg/mL to 0.15 mg/mL as well as a blank. Absorbance was measured and a linear fit was derived and used to calculate the concentration.Protein was extracted from the ground-up tubers using 0.0625M Tris-HCl, pH 6.8, and 2% SDS as previously described , in tripTotal protein was quantified by the bicinchoninic assay (BCA)  using a A calibration curve was created by using BSA at concentrations of 20–2000 µg/mL and a blank. Absorbance was measured; a linear fit was derived and used to calculate the concentration.A 50 mL tube was weighed, filled halfway with ground potato tubers, and then weighed again. The cap was replaced with a Kim-Wipe  and a rubber band. The tube was frozen at −80 °C and then lyophilized for 48 h. The tubes were weighed again after lyophilization and dry weight was recorded. Water content was calculated by subtracting the dry weight from the fresh weight, and dividing the result by the fresh weight.A total of 30 milligrams of lyophilized tubers were weighed out in triplicate from each biological replicate and extracted using a mortar and pestle with 1.5 mL of 80% ethanol in order to extract soluble sugars, centrifuged at 1600 rcf for 10 min, and incubated at 70 °C for 30 min to get rid of invertase, as previously described ,26.Sulfuric acid was slowly diluted on ice to 72%. Moreover, 200 milligrams of anthrone were dissolved in 100 mL of the diluted sulfuric acid solution, on ice, and with stirring. In glass tubes, the sample was diluted with deionized water 10-fold to 1 mL and 4 mL of the anthrone solution, and added on ice. The samples were vortexed and chilled for 30 min prior to heating. The samples were heated for 15 min in a hot water bath at 80 °C.After 15 min, the samples were cooled to room temperature and measured for absorbance at 620 nm. A calibration curve was created by using sucrose at concentrations of 5–25 µg/mL and also a blank. Absorbance was measured and a linear fit was derived and used to calculate the concentration.Potatoes set aside for the sprouting observation were kept separated by biological replicates in brown paper bags in the dark at room temperature for the entire duration of the experiment. Between 2 and 4 similarly sized tubers were used for each biological replicate (n = 20–30). Potatoes were monitored for over 120 days until all tubers reached 100% sprouting. A potato was considered to have sprouted when sprouts were over 2 mm in length as described . The numIn conclusion, this experiment attempted to suppress the three isoamylase isoforms using repeated applications of a synthesized dsRNA-lmPEI foliar spray. It was hypothesized that reduced levels of starch, reduced starch granule sizes, increased protein, and an increase in sucrose with an earlier sprouting phenotype would be observed, as was observed in a different experiment with transgenic hairpin RNAi . HoweverThe phenotype achieved in this experiment is important as it shows the initial feasibility in actively changing agricultural crop metabolism via SIGS and without transgenic modification. When considering the ease of adoption and scaling up for field agricultural uses, RNA payload protection is critical . The novAdditionally, the suppression of ISA3 in the tubers (without the suppression of ISA1 and ISA2) is significant for two reasons. First, it hints at the possibility that ISA3 may also be responsible for starch granule sizing in addition to its known roles of starch degradation and mobilization ,6. SeconSIGS has the ability to rapidly transform agriculture, with a much shorter time to the market and better acceptance than genetic transformation techniques . Targets


To analyze the effectiveness of nursing risk management in neonatal asphyxia resuscitation care and to observe and summarize the nursing measures and outcomes. n = 30) or routine care . Outcome measures included blood gas index, neonatal Apgar score, neonatal behavioral neurological assessment (NBNA) score, nursing satisfaction, and complications.  A total of 60 neonatal asphyxia cases from January 2021 to December 2021 were recruited and assigned via a random number table method at a ratio of 1 : 1 to receive either routine care plus nursing risk management  and partial pressure of carbon dioxide (PaCO2) between the two groups before care were not statistically significant (P > 0.05), while after care, PaO2 in the observation group had a higher level of PaO2 and a lower level of PaCO2 than the control group (P < 0.01). The two groups showed similar Apgar scores and NBNA scores before care (P > 0.05), while after care, routine care plus nursing risk management resulted in higher Apgar scores and NBNA scores versus routine care alone (P < 0.01). The nursing satisfaction rate in the observation group (96.67%) was significantly higher than that of the control group (73.33%) (P=0.030). Nursing risk management plus routine care was associated with a significantly lower incidence of complications (6.67%) compared to routine care (26.67%) (P=0.038).  The differences in partial pressure of oxygen (PaO Nursing risk management in neonatal asphyxia resuscitation care showed outstanding outcomes in improving neonatal blood gas index, neurological function, and Apgar score, while reducing the occurrence of complications and achieving high nursing satisfaction. As common neonatal complication , neonatan = 30) or routine care . In the observation group, there are 16 male infants and 14 female infants with an average gestation period of 38.59 ± 2.32 weeks (35–40 weeks), birth weight of 2324 g–3842 g, and mean weight of 2894.28 ± 323.68 g. There were 21 cases of mild asphyxia and 9 cases of severe asphyxia in terms of disease severity and 22 cases of vaginal delivery and 8 cases of cesarean delivery in terms of the delivery mode. In the control group, there were 17 male infants and 13 female infants with an average gestation period of 38.31 ± 2.37 weeks (36–41 weeks), birth weight of 2405 g–3850 g, and mean weight of 2915.52 ± 305.72 g. There were 21 cases of mild asphyxia and 9 cases of severe asphyxia in terms of disease severity and 22 cases of vaginal delivery and 10 cases of cesarean delivery in terms of the delivery mode. The baseline characteristics of the two groups were comparable (P > 0.05). The families of the included infants provided written informed consent after being informed of the study purpose and study procedure. This study was approved by the medical ethics committee of People's Hospital of Lujiang County, no. 59873.Sixty cases of neonatal asphyxia from January 2021 to December 2021 were recruited and assigned via a random number table method at a ratio of 1 : 1 to receive either routine care plus nursing risk management  infants who were diagnosed as neonatal asphyxia by X-ray, head ultrasound, and CT  with an In the control group, conventional nursing interventions were performed, and resuscitation was given to newborns. The ABCD process was followed to construct respiratory channels and maintain the neonate's airway open. Chest compressions and drug treatment following medical prescription were also applied, and the newborn was kept warm during the whole treatment process. The temperature of the thermal radiation table was adjusted to 32°C–34°C. A similar conventional nursing protocol was introduced to patients in the observation group.The subjects in the observation group additionally received nursing risk management. (1) A nursing risk management team for neonatal asphyxia composed of experienced head nurses, specialist nurses, and key nurses was established, and the risk factors related to the care of neonatal asphyxia were comprehensively analyzed. The nurses' cognition and management skills of neonatal asphyxia were strengthened, and resuscitation drills were conducted to improve emergency response and nursing capabilities. (2) Preresuscitation assessment: maternal, physical, and mental conditions were comprehensively monitored to keep track of maternal comorbidities, the progress of labor, and other relevant factors, with an assessment of the risk of neonatal asphyxia and preparation of resuscitators, masks, oxygen, and other emergency equipment in advance. Medical and nursing staffs were aware of their respective duties and performed accordingly to ensure smooth resuscitation. (3) Nursing during resuscitation: a 3 cm–5 cm umbilical cord should be appropriately retained during severing, to allow for subsequent umbilical vein cannulation and shorten the peripheral venous time, so as to gain time for neonatal resuscitation and increase the success rate. The newborn was kept warm on a preheated warming table, the blood and amniotic fluid on the body surface of the infant were removed, and the umbilical temperature was maintained at about 36°C-37°C. (4) Neonatal monitoring: the newborn's body temperature, pulse, and skin color were closely monitored, and any abnormalities were reported promptly to the physician for corresponding treatment. (5) Airway management: the newborn was put in a lateral position, with the head and chest appropriately padded at 15°–20°, to avoid vomit from entering the airway and to maintain respiratory flow. The respiratory tract was cleared, and back-patting and sputum suction were performed if necessary. In the event of no response from the newborn, tracheal intubation was performed for resuscitation. (6) Disinfection management: in strict accordance with aseptic operation, the ward and resuscitation room were thoroughly disinfected with good ventilation and control of the number of visitors to prevent cross-infection.The subjects received 0.4 ml per kg (body weight) of Danshen injection (diluted in 10 mL of 10% glucose injection) and 30 mg per kg (body weight) of vitamin C (diluted in 10 mL of 10% glucose injection) through an intravenous drip, with a dripping rate of 4-5 drops per minutes, twice daily. The duration of treatment was 10 days.2) and partial pressure of carbon dioxide (PaCO2), were determined with a Wofen GEM3500 automatic blood gas analyzer . (2) An Apgar score was used to assess the health status of newborns, mainly involving heart rate, breathing, muscle tone, laryngeal reflex, and skin color, with a total score of 10 points. A score of <7 points indicates the presence of asphyxia and of <4 points indicates severe asphyxia. The higher the score, the better the health status [The improvement of neonatal blood gas indexes before and after care were monitored, the changes in neonatal health status and neurological function before and after care were assessed, nursing satisfaction was obtained with a self-prepared questionnaire, and the occurrence of any complications were followed-up and recorded. (1) Blood gas indexes, including partial pressure of oxygen (PaOh status . (3) Thet-test. The test results were evaluated with 0.05 as the threshold, and a P value less than 0.05 indicated that the difference between groups was statistically significant.Data analyses were performed on the SPSS22.0 software package. The counting data are expressed as (%) and analyzed using the chi-square test, and the measurement data are expressed as (mean ± SD) and analyzed using the Student's 2 levels increased, and PaCO2 levels decreased significantly (P < 0.01). The intergroup comparison showed that the observation group had lower PaCO2 levels and higher PaO2 levels than the control group (P < 0.01)  .P > 0.05), while after care, routine care plus nursing risk management resulted in higher Apgar scores and NBNA scores versus routine care alone (P < 0.01) (The two groups showed similar Apgar scores and NBNA scores before care ( < 0.01) .P=0.030) (The nursing satisfaction rate in the observation group (96.67%) was significantly higher than that of the control group (73.33%) (P=0.030) .P=0.038) (Nursing risk management plus routine care was associated with a significantly lower incidence of complications (6.67%) compared to routine care (26.67%) (P=0.038) .Neonatal asphyxia is a state of hypoxia in which the fetus is delivered with abnormal respiratory and circulatory disorders and fails to breathe on its own. It features a high incidence and is one of the major causes of neonatal death, cerebral palsy, and mental retardation . ResearcIn addition to necessary clinical treatment and resuscitation, nursing interventions are essential to reduce mortality and enhance the prognosis of neonatal asphyxia. Conventional care of neonatal asphyxia mainly focuses on disease observation and resuscitation, which lacks foresight and has a poor emergency response. As a new nursing management model, nursing risk management accurately and effectively identifies potential risk factors and uncertainties in nursing work and formulates relevant countermeasures in advance, which reduces the damage to the children and ensures nursing safety. Nursing risk management transforms passive care into active care. The results of the present study showed that risk nursing management was associated with significantly better blood gas indices versus routine care, indicating that nursing risk management improved the blood gas of the children. Infection is the most common complication of neonatal asphyxia, which is attributed to improper aseptic operation and inadequate disinfection. Therefore, disinfection management should be strengthened for infection prevention to avoid cross-infection, pulmonary infection, and other related complications. As neonatal asphyxia is often accompanied by hypothermia, nursing staff should enhance their understanding of its risk factors and strengthen thermal care. Early risk management and proper care reduce the incidence of ischemic-hypoxic encephalopathy and improve the neurological function of children. In the present study, risk nursing management resulted in significantly higher Apgar scores in patients and a lower incidence of complications versus routine care, suggesting that the application of nursing risk management improves neonatal health status and reduces the damage to neurological function. Nursing risk management is patient-centered and proactively assesses potential risk factors based on the actual clinical situation of the patient to formulate corresponding contingency plans and specific treatment measures to restrain all potential risks, which greatly improves the quality of care. Previous studies have reported that risk management could reduce nurse-patient disputes, enhance the prognosis of the child, and promote nursing satisfaction. In the present study, the nursing satisfaction of the observation group was higher than that of the control group, indicating that the families of these infants were satisfied with this nursing method.Treatment with Danshen injection and vitamin C for neonatal asphyxia improves the recovery, reduces sequelae, shortens the duration of treatment, and decreases the frequency and dosage of continuous antispasmodics. Vitamin C can remove excessive free radicals produced during hypoxic brain damage, and Danshen injection can improve microcirculation, enhance brain tissue metabolism and regulation, and repair damaged brain cells, thereby improving the therapeutic effect and reducing sequelae. The current issues in risk nursing management include the enhancement of the training of asphyxia resuscitation personnel and further perfection of evaluation indicators of successful resuscitation of asphyxiated newborns.Nursing risk management in neonatal asphyxia resuscitation care showed outstanding outcomes in improving neonatal blood gas index, neurological function, and Apgar score, while reducing the occurrence of complications and achieving high nursing satisfaction. The limitation of this study is the absence of long-term follow-up of children and regular monitoring of their intelligence to support the effectiveness of this care strategy. Future studies with long-term follow-up are required to provide more reliable data for clinical references.