The signal pathway of actin remodeling, including LIM-kinase 1 (LIMK1) and its substrate cofilin, regulates multiple processes in neurons of vertebrates and invertebrates. Drosophila melanogaster is widely used as a model object for studying mechanisms of memory formation, storage, retrieval and forgetting. Previously, active forgetting in Drosophila was investigated in the standard Pavlovian olfactory conditioning paradigm. The role of specific dopaminergic neurons (DAN) and components of the actin remodeling pathway in different forms of forgetting was shown. In our research, we investigated the role of LIMK1 in Drosophila memory and forgetting in the conditioned courtship suppression paradigm (CCSP). In the Drosophila brain, LIMK1 and p-cofilin levels appeared to be low in specific neuropil structures, including the mushroom body (MB) lobes and the central complex. At the same time, LIMK1 was observed in cell bodies, such as DAN clusters regulating memory formation in CCSP. We applied GAL4 × UAS binary system to induce limk1 RNA interference in different types of neurons. The hybrid strain with limk1 interference in MB lobes and glia showed an increase in 3-h short-term memory (STM), without significant effects on long-term memory. limk1 interference in cholinergic neurons (CHN) impaired STM, while its interference in DAN and serotoninergic neurons (SRN) also dramatically impaired the flies’ learning ability. By contrast, limk1 interference in fruitless neurons (FRN) resulted in increased 15–60 min STM, indicating a possible LIMK1 role in active forgetting. Males with limk1 interference in CHN and FRN also showed the opposite trends of courtship song parameters changes. Thus, LIMK1 effects on the Drosophila male memory and courtship song appeared to depend on the neuronal type or brain structure. Memory formation and forgetting serve as the basis of behavioralplasticity. Whereas memory is a specific process ofinformation acquisition, storage and retrieval by the nervoussystem, active forgetting is defined as “a mechanism or seriesof mechanisms to remove memories that become unused”. Associative memory formation andactive forgetting occur in both mammals and invertebrates,including Drosophila melanogaster , whichis a well-known object of classical genetics. Having a shortlife cycle and relatively simple nervous system, the fruit flymakes it easy to perform genetic analysis of the molecularbasis of behavioral and cognitive processes.There are several experimental techniques to form associativememory in Drosophila, including short-term memory(STM) and protein synthesis-dependent long-term me-mory(LTM). The most widely used technique is classical Pavlovianlearning with negative electroshock reinforcement,or olfactory aversive learning (OAVL), which revealed genesresponsible for different types of memory .More natural is conditioned courtship suppression paradigm(CCSP) .GAL4 × UAS binary expression system  is usedto study the effects of specific genes on memory processes.The fine neural organization of the mushroom bodies (MB),a principle structure responsible for associative olfactorylearning in Drosophila, was evaluated in detail. The MB outputneurons (MBON) are the main effectors of MB, whereasspecific clusters of dopaminergic neurons (DAN) regulate theactivity of MB – MBON synaptic contacts . Among them are aSP13 DAN of the protocerebralanterior medial cluster (PAM), which innervate γ5 area ofMB, playing a crucial role in CCSP learning and memory.The molecular and neural mechanisms of active forgettingimplicate the activity of DAN and Rac1-dependent signalpathways . Small GTPases of the Rho family,including Rho and Rac, regulate neuronal actin polymerizationduring the Drosophila nervous system development. Rho viaits effector ROCK or Rac/Cdc42 via its effector Pak activateLIM-kinase 1 (LIMK1), which phosphorylates Drosophilacofilin (twinstar) protein, blocking its actin-depolymerizationactivity and inhibiting axon growth. Rac also acts through Pakindependentpathway to antagonize LIMK1 and promote axongrowth . In addition to its role in neurogenesis,Rac is crucial for both interference-induced and passiveforgetting in OAVL paradigm. PAK/LIMK1/cofilin pathwayprobably acts downstream Rac1 . Forgettingspecific types of memory depends on different signal proteins.Forgetting in OAVL paradigm is caused by several DANof the protocerebral posterior lateral 1 (PPL1) cluster, whichinnervates some MB structures, such as pedunculus, lowerand upper stalk. Memory acquisition and forgetting are regulatedby different dopamine receptors, dDA1 and DAMBrespectively . Coincidence of conditionedand unconditioned stimuli creates a memory trace in MBON-γ2αʹ1, probably inhibiting the MB > MBON-γ2αʹ1 synapses.The unconditioned stimulus alone activates DAN-γ2αʹ1,which in turn disinhibit MB > MBON-γ2αʹ1 synapses andcause forgetting . DAN that innervate theMB ααʹ tip induce the interference-based forgetting throughthe scaffold protein Scribble, binding together Rac1, PAK3and cofilin .Whereas multiple data prove the importance of DAN andactin-remodeling signal pathway for forgetting in OAVL paradigm,there is virtually no data for molecular mechanisms ofmemory decay in CCSP. Effects of LIMK1-dependent signalcascade on CCSP learning and memory were firstly shown forthe temperature-sensitive mutant agn ts3, with LIMK1 increasein the adult brain compared to the wild type Canton-S (CS).Temperature rise leads to a decrease in agn ts3 LIMK1 level,simultaneously restoring its learning ability and 3 h memory,which are drastically impaired in the norm . agn ts3 has multiple polymorphisms within and nearlimk1 gene, as well as a changed profile of microRNA expression,and can serve as a model object for Williams syndrome. Thetemporal profile of STM learning index (LI) was assayedin CCSP for agn ts3, as well as for the wild-type strains withlimk1 polymorphisms, CS and Oregon R. Only CS was able tolearn and store memory up to 24 h .The behavioral effects of LIMK1 changes in agn ts3 do notgive information about specific cell types, where LIMK1 canbe involved in learning and memory. In this study, we performedthe analysis of memory decay for several DrosophilaGAL4 × UAS strains with neuronal type-specific limk1 RNAinterference. LIMK1 distribution in the Drosophila brainstructures was studied in detail using confocal microscopy. Theeffect of limk1 interference on fly memory ability dependedon both neural type and memory form. LIMK1 also appearedto be involved in regulation of male courtship song: limk1interference in different neuronal types specifically affectedsome song parameters.Drosophila strains. Fly strains were provided by the ResearchCenter “Biocollection of Pavlov Institute of Physiology RASfor the study of integrative mechanisms of the nervous andvisceral systems”. The strain numbers (#) are given in accordancewith the Research Center and Bloomington DrosophilaStock Center, USA . The following strainswere used:1. Сanton-S (СS) – the wild-type strain with limk1 polymorphisms.2. agn ts3 – temperature-sensitive mutation on CS geneticbackgroundwith limk1 polymorphisms, characterized bylearning and memory defects.3. Strains expressing GAL4 in specific neuronal types:#6794: w[*]; P{w[+mC]=nrv2-GAL4.S}8 P{w[+mC]=UAS-GFP.S65T}eg[T10]. GAL4 and green fluorescentprotein (GFP) are expressed in nervous system under Nrv2regulatory element;#6793: w[*]; P{w[+mC]=ChAT-GAL4.7.4}19BP{w[+mC]=UAS-GFP.S65T}Myo31DF[T2]. GAL4 andGFP are expressed in cholinergic neurons (CHN) underChAT and VAChT regulatory elements;#7009: w[1118]; P{w[+mC]=Ddc-GAL4.L}Lmpt[4.36].GAL4 is expressed in dopaminergic (DAN) and serotoninergic(SRN) neurons;#30027: w[1118]; P{w[+mW.hs]=GawB}fru[NP0021].GAL4 is expressed in fruitless neurons regulating matingbehavior.4. Act-GAL4: w[1118]; P{w[+mC]=} 25FO1/CyO, y[+];Canton-S background. GAL4 is expressed in the wholebody under actin promoter.5. Strains with UAS-dependent limk1 suppression:#26294: y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02063}attP2. The strain expresses interfering RNA against limk1(RNAi) under UAS .#36303: y[1] v[1]; P{y[+t7.7]=CaryP}attP2. The controlstrain with genetic background identic to that for #26294,but lacking RNAi (limk1“+”).6. Strains with GFP gene regulated by UAS:#32186: w[*]; P{y[+t7.7] w[+mC]=10XUAS-IVS-mCD8::GFP}attP40.#32202: w[*]; P{y[+t7.7] w[+mC]=10XUAS-IVS-GFPWPRE}attP2.To induce limk1 RNA interference in specific neuronaltypes, a strain carrying GAL4 activator expressed under tissue-specific promoter was crossed to UAS strain #26294. Theсross product of a GAL4 strain and #36303 strain served asa control.Flies were raised on the standard yeast–raisin medium at25 ± 0.5 °C and a 12:12 daily illumination cycle. For behavioraltests, experimental males were collected without anesthesiaand kept individually. 5–6-day-old males were used in experiments.Females (CS) were collected as virgins and broughttogether with CS males for fertilization in CCSP one daybefore experiment.Antibodies. Primary antibodies: Rat anti-LIMK1 multi-specificmonoclonal ; mouse anti-Drosophila cysteine string protein (CSP);rabbit anti-Drosophila tyrosine hydroxylase (TH) ; rabbit anti-GFP .Secondary antibodies: Goat anti-mouse Alexa Fluor 488, donkey-anti-rat Alexa Fluor 594, goat anti-rabbit AlexaFluor 633 .RNA extraction and RT-PCR analysis of limk1 expression.The level of limk1 expression was assayed usingsemi-quantitative PCR in complex with reverse transcription(RT-PCR). Flies were anesthetized by freezing. 10 male fliesor 70 male heads were homogenized in 300 μl TRI reagent. Total RNA was extracted from homogenatesaccording to the manufacturer’s protocol. The quality ofRNA was checked by 1.5 % agarose gel electrophoresis. 1 μgRNA was reverse-transcribed by MMLV reverse transcriptase according to the manufacturers’ protocol,using random hexamer primers and RNAse inhibitor . Semi-quantitative PCR was performed on a StepOnePlus  using qPCRmixHSSYBR+LowROX  containing direct andreverse primers (0.5 mM each). Baseline and cycle thresholdvalues were determined by automatic analysis using StepOnesoftware v2.3 . rpl32 transcriptwas used as an internal control. The predesigned limk1 primers were used to bind all five limk1 cDNA isoforms,both premature and mature forms, as primers do notspan the exon-intron borders. The relative limk1 transcriptlevel was calculated using the comparative ΔΔCt method. Thenumber of biological replicates (independent RNA extractionswith reverse transcription) was 3–5, the number of technicalreplicates was 3.The primer sequences were the following:rpl32:Forward: 5′-TATGCTAAGCTGTCGCACAAATGGC-3′Reverse: 5′-GTTCTGCATGAGCAGGACCTCCA-3′limk1:Forward: 5′-GTGAACGGCACACCAGTTAGT-3′Reverse: 5′-ACTTGCACCGGATCATGCTC-3′PCR parameters:1. 1 cycle: 95 °С – 5 min.2. 45 cycles. 95 °С – 20 s, 60 °С – 20 s, 72 °С – 20 s, 77 °С –15 s (detection).3. Melting curve: 95 °С – 15 s, 60 °С – 1 min, 60–95 °С.Immunofluorescent staining of Drosophila brains.5–6-day-old imago males were anesthetized by freezing. Thebrains were prepared in PBS buffer (pH 7.5) using needlesharptweezers , fixed in 4 % paraformaldehydein PBS for 1 h at RT and stained according to , without a freezing stage. Antibodies were dilutedin PBT  as 1:200, foranti-CSP – 1:20. Previously, for better staining of brains, weincreased the time of incubation with primary antibodies upto 5 days . Here, the incubation wasperformed at 4 °С for 3 days (with primary antibodies) orovernight (with secondary antibodies). Brains were mountedwith Vectashield mounting medium containing DAPI .Protein distribution analysis in the brain by confocalmicroscopy. Brains were scanned frontally using laser scanningconfocal microscopy .Scanning was performed using X63 objective at differentdepths (z-step 2 μm). Images were analyzed using Fiji software.The brain structures were visually mapped using theDrosophila brain online atlas . To measurethe average level of LIMK1 inside the brain structures, theaverage signal intensity was measured in three small squareareas (~10 × 10 μm) within each of the structures. The averagevalues were obtained and normalized to the average structureintensity for the given brain. Colocalization Threshold analysis was performed to measure co-localization of LIMK1 withneurospecific markers. To prepare figures, auto contrast functionwas used for each optical slice.Learning and courtship suppression tests in Drosophilamales. Flies learning and STM were estimated in CCSP, asdescribed in . In the case of long termmemory (LTM), learning was performed by placing flies infood-containing glasses  for5 h . Courtship index (CI) and learningindex (LI) were estimated at the following time pointsafter learning: for short-term memory (STM) analysis: 0 min(learning), 3 h; for STM decay analysis: 15, 30, 60 min,24 h; for LTM analysis: 0 min, 2 days, 8 days. In all groups,naive males (without mating experience) served as a controlto calculate LI:where CIN is the middle CI for naive males, and CIT is themiddle CI for males after training. The naive and trainedmales were the same age. The decrease in LI compared toLI (0 min) was considered a time-dependent memory decay.The decrease in LI for a mutant strain compared to that for thewild-type strain CS was considered a strain-specific impairmentof learning or memory.Courtship song analysis. The 5-day-old imago malecourtship song was recorded as in . A naive male of the studied line and a fertilizedfemale (CS) were placed together in a Perplex chamber witha latticed bottom on top of a microphone. The chamber wasplaced in a foam box in a soundproof room. The sounds wererecorded for 5 min using Audacity software . The sound signals were filtered to excludenoises, obtaining signals within 100–800 Hz. The level ofnoise was decreased using a standard Audacity plugin. Thesoftware Drosophila courtship song analysis (DCSA)  was used to automatically detect pulse and sinesong components.The results of analysis were manually edited. The meanvalues of the song parameters were calculated for each fly.The following parameters were estimated: pulse song index, pulse song initiation frequency, sine song index , sinesong frequency , interpulse interval ,period of song pulse train , intertrain interval ,train duration (TrainDur), pulse number in train (PulseN), sinesong duration , sine song amplitude ,IPI variance (Var(IPI), ms2). Per is the time between the startsof the neighboring trains. ITI is the time between the end ofthe previous and the start of the next train.Statistical analysis. Analysis of LIMK1 mRNA level wasperformed using two-sided t-test, Social Science Statisticonline resource ( p < 0.05). Analysis of LI and courtship songparameters was performed using two-sided randomizationtest at significance level α of 0.05 (n = 20), using DrosophilaCourtship Lite software , with 10000 iterations. The program is freelyavailable from the author upon request. Randomization testwas reported to be better for LI comparison than t-test orsome nonparametric tests . Courtshipsong parameters were also analyzed using two-sidedMann–Whitney U-test. Python 3 scripts were used to drawthe box plots charts.limk1 RNA level in Drosophila UAS × GAL4 hybridsTo check that GAL4 really induces limk1 RNA interferencein 26294 strain, we compared limk1 RNA level in the UAS (f)> GAL4 (m) hybrids. Females with and without transgenicRNAi for limk1 suppression were crossed to Act-GAL4 males, expressingGAL4 in the whole body. The level of total limk1 RNA wasapproximately 2-fold lower in the hybrid with limk1 interference.These data confirmed the efficiency of RNAi-dependentlimk1 suppression in 26294 strain (limk1-KD) upon its activationby GAL4. At the same time, there were no differences forlimk1-KD > 6794 and limk1“+” > 6794, where RNA expressionwas measured in heads and was regulated by neuronaltype-specific GAL4 . Thus, limk1 RNA differencesafter neural type-specific limk1 RNA interference might belocal or too low to be detected in the whole Drosophila heads.LIMK1 distribution in the Drosophila brainWhen studying LIMK1 distribution, we focused on thecentralpart of the Drosophila brain, without the optic lobes(OL), mainly at the level of the superior medial protocerebrum(SMP) and gamma-lobes (γL) of MB. Here the PAMclusters of DAN are located , responsiblefor the Drosophila courtship learning and memory . Additionally, the area including the central complex(CC) and calyx  surrounded by Kenyon cells (KC)was studied. The CSP-positive neuropil structures and tyrosinehydroxylase (TH)-positive DAN cell bodies and processesserved as landmarks in describing the LIMK1 distribution.The following description is given for the wild-type strainCS 1.https://vavilovj-icg.ru/download/pict-2023-27/appx9.pdfSupplementary Materials are available in the online version of the paper:The distribution of DAN clusters corresponded to thatdescribed in . PAM clusters were clearlyobserved near SMP, with processes extending towards thecentral part of the brain. The processes formed glomerularstructures around the MB horizontal lobes , probablybeing the synaptic endings innervating the correspondareas. The structure #3 was located above γL, the structures#5 and 8 – in the β′L area, the commissure #9 was seen in thecentral part of the brain. TH signal was relatively low in βL. PPL1, PPM2 and other DAN clusterswere observed around Cal, sending their processes to the differentbrain areas .LIMK1 was concentrated in the neuropil structures of theanterior part of the brain, such as SMP and AL. The LIMK1-positive granularity was observed inside SMP, between the β′Ltips (#8) and around the ellipsoid body (EB) of CC .LIMK1 level was also high in thin tissue layers adjacentto neuropil and some neural tracts, such as #12 around thegreat commissure, #13 around wedge (W) and #15 near esophagus(ES), morphologically resembling glia . LIMK1 signal was lower in cell bodies of the neuronssurrounding AL (ALCB), probably being the cell bodies of theprojection neurons, as well as in KC surrounding Cal. Here,LIMK1 was mainly concentrated in the cytoplasm, beyondthe nuclei. LIMK1 level was significantly decreased in all theMB lobes and pedunculus (Ped), as well as in the CC structures,whereas in Cal and the protocerebral bridge (PB) it wasrelatively high . LIMK1 and TH colocalizationwas observed in SMP, AL, Cal, the TH-positivecells and processes, and in glomerular densities, such as #3,5 and 6 .To check that the antibody specifically binds to LIMK1, thedistribution of LIMK1 main product p-cofilin was assayed inCS. The pattern of p-cofilin distribution was generally similarto that for LIMK1 . The level of p-cofilinwas low in MB  and CC . In contrast to LIMK1, p-cofilin wasmainly concentrated in the cell nuclei in the peripheral area ofthe fly brain, such as Kenyon cells around Cal, as well as in PB,subesophageal ganglion (SEG), and cell bodies surrounding AL. p-Cofilin was also localized in diffuse layers within thebrain structures, such as EB, probably formed by glia. Thep- cofilin-enriched cells were found in SEG, forming structureswith two glomerular branches (*), and around CC structures,fan-shaped body (FB) and EB (**).Several GAL4 activators were used to initiate limk1 RNAinterference. Both 6793 and 6794 strains specifically expressthe green fluorescent protein (GFP) under GAL4 promoter.In strain 6794, GAL4 was reported to be expressed in OL,thoracic ganglion, different nerves, and cortex glia . In 6794 > limk1-KDhybrid, GAL4-driven GFP expression was detected in glialikecells, surrounding the neuropil structures, such as AL,SMP, CC and its parts, as well as in the MB lobes, Ped andsome KC . GFP level was higher in αLand βL compared to α′L, β′L and γL. The signal was lowerin Cal and virtually absent in most neuropil structures, suchas AL, SMP, CC and others. Thus, limk1 interference shouldoccur in Cal and some glia cells, where the levels of bothGAL4 and LIMK1 were relatively high. Similar distributionwas observed in the control 6794 > limk1“+” strain . In the strain 6793, GAL4 is expressedin cholinergic neurons (CHN), with GFP signal in OL, ALwith the surrounding interneurons, the parts of CC, the greatcommissure (GC), Cal and the mechanosensory area of SEG. In the strains 6793 > limk1- KDand 6793 > limk1“+” , we observeda strong GFP signal in cell bodies surrounding SMPand AL, as well as in some KC, several neuropil structures, and GC. In all the studied strains, LIMK1distribution character appeared to be similar to that in CS.To check that GAL4 is active in 7009 and 30027 strains,we crossed them to strains expressing GFP under GAL4 promoter.In 7009 > 32186 hybrid, we observed a prominent GFPsignal in cell clusters near SMP, morphologically similar tothe TH-positive PAM clusters. Some cells might be SRN, butthey constitute the minority of the observed neurons in thisarea . The processesof PAM neurons extended to the horizontal MB lobes, includingγL, and the densely innervated β′L tip (#5) connected by a commissure, and to a much lesser extent the βL tip . EB was surrounded by the GFP-positive processesextending from different parts of the brain. The GFPpositiveDAN around Cal were also observed. Hence, GAL4activator of 7009 should suppress limk1 inside DAN, includingPAM neurons, which regulate memory storage in CCSP. Thefruitless-positive neurons (FRN) are responsible for matingbehavior. In 30027 > 32202 hybrid, we observed GFP insome KC, in the cell bodies located near SMP and AL, andglomerular structure, forming a ring-like structure around Ped. Similar structures were describedin . The distribution of LIMK1 in the hybridstrains with and without limk1 knockdown in the above neuronswas similar to that in CS .The normalized intensity of LIMK1 signal was calculatedfor several brain structures . The LIMK1 relative levelsin specific structures were very similar for the CS brain andthe average brain of all the strains. The biggest differenceswere observed for the TH-positive glomerular structure #6(TH+(6)), which is possibly responsible for memory formationin CCSP. In the average brain, ALCB had the normalizedLIMK1 level about 1. Compared to them, AL, SMP andTH+(6) structures had the higher LIMK1 level, whereas theMB lobes, EB and Ped had the lower LIMK1 level. In agn ts3,AL and ALCB had the higher LIMK1 level compared to CS,whereas most of the rest studies structures had lower LIMK1level. This corresponds to more contrast LIMK1 staining inagn ts3 relative to CS . There were no prominentdifferences after limk1 knockout, except for severalstructures with minor changes. The interstrain differencesmight be local or beyond the resolution of the method.3 h STM differs in hybridswith and without limk1 interference3 h STM was estimated for limk1-KD (f ) > 6794 (m) and thecontrol limk1“+” (f ) > 6794 (m) hybrids. In both cases, weobserved the decrease of courtship index (CI) after learning,with its partial recovery after 3 h. The box plot height wasminimal for CS and rather big for UAS × GAL4 hybrids,showing that the value of courtship suppression significantlyvaried for individual flies. All strains were capable to learn inCCSP, with learning index LI (0 h) immediately after trainingof about 60–70 % . The CS LI was still high after 3 h,indicating STM preservation, in agreement with . The strain with limk1 interference also preservedSTM: although its LI (3 h) was only about 20 %, it did notstatistically differ from that for CS, as well as from LI (0 h). Inthe control strain, LI (3 h) decreased compared to LI (0 h) anddid not differ from zero, indicating the impaired STM. Thus,while 3 h memory storage or retrieval was impaired in thecontrol strain, limk1 interference seems to improve 3 h STM.At the same time, it did not affect the impaired 8 day LTM,with only minor positive effect on 2 day LTM .Neuron type-specific limk1 interferencedifferentially affects STM dynamicsTo investigate the dynamics of STM decay in different strainswith limk1 interference, we performed LI analysis immediatelyand 24 h after learning . To exclude the possible effectof eye color on learning and memory abilities, we appliedGAL4 (f ) > UAS (m) crossing scheme, where both the strainwith limk1 knockdown and the control strain had the samewild-type eye color. For 6794 activator (MB and glia), thecontrol strain showed nearly the same LI within 24 h, whereasthe strain with limk1 interference demonstrated a steeperforgetting curve. Hence, 6794 > limk1-KD showed high LIafter learning, but seemed to increase the speed of memoryforgetting on the interval 0–30 min. limk1 knockdown in CHN(6793) was associated with significant decrease of LI within60 min after training.For DAN and SRN activator (7009), both the strain withlimk1 interference and the control strain showed nearly thesame dynamics of STM decay, except for 30–60 min period.limk1 interference was associated with a dramatic defect onlearning: LI did not differ from zero. LI (24 h) was negativein both hybrids, possibly being the effect of sensitization:males did not suppress the courtship activity but courted moreactively some time after training. For FRN activator (30027),the effect of limk1 knockdown was the opposite to that ofMB/glia and CHN activators: limk1 interference decreasedthe speed of forgetting, and LI (30 min) did not differ fromzero. Thus, the effect of limk1 interference on STM dynamicsappeared to depend on the neuronal type.LIMK1 interference in CHN and FRN neuronsdifferentially affects courtship song parametersFinally, we studied the influence of limk1 interference on themale courtship song parameters. The hybrids with CHN andFRN drivers were studied . There were no interstraindifferences in interpulse interval (IPI), the species- and population-specific parameter , and IPI variance(Var(IPI)), the marker of neurodegenerative processes. limk1 interference in CHN(6793) decreased the pulse song index and frequency , increasing the mean period (Per), intertrain interval (ITI),train duration (TrainDur), sine song duration (Sdur) and trainpulse number (PulseN). On the contrary, in the strain with FRNactivator (30027), limk1 interference resulted in PFr increase,as well as Per, ITI, SInd and SDur decrease. limk1 knockdownby two different activators had the opposite effects on PInd,PFr, Per, ITI and SDur, leveling the initial differences betweenSInd, TrainDur and PulseN. Thus, limk1 interference in CHNseemed to decrease the rate of switching from the singingmode to silence mode and back, resulting in longer trains andITI, while limk1 interference in FRN neurons generally hadthe opposite effect.In mammals, LIMK1- and cofilin-dependent actin remodelingis widely involved in regulation of synaptic processes,such as exocytosis, receptor trafficking and remodeling ofdendritic spines. These processes underlay long-term potentiation(LTP), long-term depression (LTD) and different forms ofmemory. LIMK1 also affects gene expression through CREBand LTM formation. Deregulation of LIMK1-dependentactin remodeling is involved in multiple pathologies, such asAlzheimer’s and Parkinson’s diseases, Williams syndrome,schizophrenia, and autism .In mature neurons, actin is enriched in both pre- and postsynapticstructures, such as dendritic spines, regulated byRho signaling pathway. The action of LIMK1 on actin polymerizationand memory processes is rather complex, beingdependent on the mode of LIMK1 regulation (transient orlong-term overexpression) and cofilin level. While the activecofilin destabilizes fibrillar actin, in high concentrations itincreases actin polymerization and nucleates actin filamentsin dendritic spines during long-term potentiation . Thus, it is hard to predict the integral effect of LIMK1 and cofilin activity on memory processes.It was crucial to check the behavioral effects of Drosophilalimk1 suppression in specific types of neurons.Using paraffin section staining, both LIMK1 and its productp-cofilin were shown to be homogeneously distributed in thebrain neuropil, with maximum level in CC . Our results of the confocal microscopy analysis gavea different picture of LIMK1 distribution, quite similar for allthe studied strains. We have shown a specific LIMK1 decreasein MB, which is responsible for associative learning, as well asin CC, involved in higher movement control . This puts under question the role of LIMK1 inthe aforementioned processes. However, LIMK1 was presentin the cell bodies and processes of DAN, which interact withMB and CC  and regulate memory andforgetting. The observed p-cofilin distribution resembled thisfor LIMK1: its level was low in the MB lobes and CC. The lowlevel of p-cofilin in the MB lobes had been previously shown . In contrast to LIMK1, p-cofilin level waslow in Cal formed by PN and KC terminals and high in cellnuclei. The latter corresponds to its functioning in the cell, ascofilin phosphorylation is necessary for its translocation intothe nucleus .The effectiveness of limk1 suppression at the RNA level wasconfirmed using Act-GAL4 activator in the whole Drosophilabody. GAL4 was also active in specific brain areas of the correspondingstrains. However, we failed to quantitatively checkthe changes of limk1 expression in Drosophila UAS × GAL4hybrids with neuronal-specific GAL4 expression. The decreasein LIMK1 level might be local or too small. limk1interference might also induce the compensatory activationof LIMK1 translation.To study limk1 knockdown effects on memory, we usedCCSP modification applied by :training was performed with the mated female. In this case,courtship learning results from the rise of sensitivity to theantiaphrodisiac cis-vaccenyl acetate (cVA) due to unsuccessfulcourtship. cVA is not required for learning, being necessaryfor memory performance. aSP13 DAN, which innervate thefru-positive tip of γL, are necessary and sufficient for courtshiplearning . 24 h memory consolidationrequires the prolonged aSP13 stimulation and Orb2 dimerizationin some γ neurons . α/β neurons areinvolved in LTM processes . Hence, other DAN innervating α/βL of MB,including PAM and PPL1 cells , may alsobe involved in LTM.The behavioral differences were observed after limk1 interference,e. g., the restoration of 3 h STM for limk1-KD >6794 strain. GAL4 drivers themselves affected memoryabilities,which were generally decreased compared to CS.The drivers also significantly affected the forgetting curves.Thus, we studied the effects of limk1 interference relative toa control strain with the same GAL4 driver. We applied twodifferent crossing schemes – UAS (f) > 6794 (m)  and reverse . In the first case, the control UAS > limk1“+”hybrid had bright eyes due to v[1] recessive allele, in contrastto UAS > limk1-KD hybrid with the wild type dark red eyes.The observed 3 h STM differences are unlikely to be associatedwith the differences in eyes pigmentation, as v[1] fliesshowed a normal 3 h STM and 2 day LTM in CCSP , while both forms of memory were impaired in thecontrol strain. However, memory retention depends on parentaffect, with some paternal epigenetic factors affecting STMstrength . For 6794 > limk1-KD strain,we did not see STM difference from the control strain, thoughlearning ability slightly increased after limk1 knockdown . Thus, when studying LIMK1 effects on learning andmemory, it is necessary to consider the crossing direction.Acetylcholine is the major excitatory neurotransmitter inDrosophila. Among CNH are: PN forming synapses on KCof MB , the MB intrinsic neurons thatare responsible for olfactory memory, expressing ChAT andVAChT , and the α/β core neurons requiredfor LTM consolidation . In the hybridswith 6793 driver (GAL4 expressed in CHN), GFP level wasspecifically high in α/βL compared to the other MB lobes.Here, limk1 interference resulted in faster STM forgetting.This contradicts the cofilin role in active forgetting shown inOAVL, where cofilin was proposed to be regulated by LIMK1. The involvement of LIMK1 and cofilinin forgetting may occur locally, within specific neuronalpopulations or synaptic terminals. At the same time, LIMK1may be crucial for memory storage and retrieval in CCSP.The glutamatergic MBON M6 neurons serve for STM output:aSP13 DAN prolongs potentiation of γL – M6 synapses . Some cholinergic MBON appeared to regulatethe Drosophila visual appetitive memory .As the extrinsic MB cells responsible for CCSP memory weresimilar to those used for appetitive memory , the decrease in 60 min STM might occur due to limk1suppression in some of these neurons.The hybrids with 7009 driver (DAN and SRN) showedgenerally low CI values and negative LI values 24 h afterlearning. Males of these strains had pale pink eyes because ofdefects of eyes pigmentation, due to non-complete w[1118]rescue. w[1118] males demonstrated low courtship activityand success, presumably due to some defects of sexual developmentand maturation . However, thecontrol 7009 > limk1“+” strain had normal LI up to 60 minafter training. limk1 knockdown by 7009 driver was associatedwith dramatic defects of learning and memory: LI just aftertraining did not statistically differ from zero. Thus, LIMK1-dependent signaling in DAN and SRN seems to be importantfor learning and memory in CCSP.limk1 knockdown by 30027 driver (FRN) decreased theforgetting rate in the time interval 30–60 min. This correspondsto the role of actin-remodeling pathway in forgettingin OAVL paradigm . LI of the controlstrain did not differ from zero starting from 30 min afterlearning, while limk1 knockdown increased it. In males, FRNare responsible for courtship behavior. There are ~1500 FRNin the Drosophila brain, including sensory organs, lateralhorn, lateral protocerebrum, SMP arch and motor controlcenters. Together they provide multisensory integration toregulate the male courtship process . Some CHN and DAN are also Fru-positive, suchas ~300 γL neurons and aSP13 DAN located in SMP, whichregulates courtship learning and memory. The activity of frugene was reported to decrease upon LTM formation in CCSP. Hence, suppression of some FRN activitymay be associated with memory prolongation and consolidation.In addition to memory processes, limk1 interference affectedsome parameters of the male courtship song. As well as forcourtship memory, we observed the opposite functional effectsof limk1 knockdown in FRN and CHN. FRN of the P1 classinitiate Drosophila courtship behavior and trigger courtshipsong. pIP10 neurons possibly convey the P1 signal to thoracicdPR1 and vPR6 neurons, proposed to be the parts of a centralpattern generator (CPG), which defines the time and shape ofthe pulse song. vPR6 possibly encode IPI . Pulse and sine CPG either contain FRN or interactwith them. As sine and pulse song normally do not overlap,the mutually inhibitory mechanisms must exist, switching between quasilinear and relaxation modes of oscillation forsine and pulse song, respectively. Some descending interneuronsmay control the type of the song, while the others triggersinging or terminate the song .Indeed, we observed the opposite changes of PInd/PFr andSInd/SDur upon limk1 interference in CHN and FRN, movingthe balance toward the sine and pulse song, respectively. Theincrease in PFr after limk1 knockdown in FRN might indicatethe negative role of LIMK1-dependent signaling on activityof the pulse CPG or the upstream brain centers, which switchthem from active to silent mode. СС is important for control ofstability of pacemakers, which regulate the rhythmic structureof courtship song. PB destruction leads to sound signal distortions,FB and EB destruction additionally decreases sine andpulse trains . CC includes a large numberof neuronal types, such as CHN, DAN, SRN, and others. CHNare present in FB, EB, No and PB ,similarly to what we observed in our research. Hence, theyprobably play some role in regulation of male singing. Theopposite effects of limk1 interference in CHN and FRN mayindicate a specific role of LIMK1 in courtship controllingnetwork, whereas the other parts of the brain possibly havea total antagonistic effects on its activity. Alternatively, CHNand FRN fru neurons may differ in some aspects of regulationof LIMK1-dependent signaling pathway.In summary, we have shown that effects of limk1 interferencein Drosophila male courtship memory and song depend onboth the neuronal type and specific behavioral parameter.limk1 interference in CHN and FRN had generally oppositeeffects, whereas its suppression in DAN and SRN impaired theflies’ ability to learn. Using activator strains with a narrowerpattern of GAL4 expression would help to better localize thebrain structures, where LIMK1 regulates memory and forgettingin CCSP. Among such putative structures are γL andaSP13 DAN innervating γ5 area, as well as other DAN participatingin memory formation, consolidation and retrieval.Studying the behavioral consequences of limk1 overexpressionin different brain areas will complement the estimates of theeffects of its suppression. The above investigation should alsofocus on LIMK1 partner proteins, such as cofilin in its activeand phosphorylated form.The authors declare no conflict of interest.Abe H., Nagaoka R., Obinata T. Cytoplasmic localization and nucleartransport of cofilin in cultured myotubes. Exp. Cell Res. 1993;206(1):1-10. DOI 10.1006/excr.1993.1113.Abe T., Yamazaki D., Murakami S., Hiroi M., Nitta Y., Maeyama Y.,Tabata T. 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To date, etravirine (ETR) is the only NNRTI indicated in people living with HIV (PLHIV) after virologic failure (VF) to first-generation NNRTIs. Data about susceptibility to ETR, doravirine (DOR) and rilpivirine (RPV) after VF in PLHIV from Latin American countries are scarce. The aim of this study is to determine the prevalence of resistance to ETR, DOR, and RPV in PLHIV who experienced VF to a first-line NNRTI-based regimen, and factors associated with high-level resistance (HLR) to ETR.Retrospective cohort study. Prevalence of resistance to ETR, DOR and RPV was assessed by analyzing genotyping tests of adult PLHIV who experienced VF to a first-line NNRTI-based regimen, in Buenos Aires, Argentina (2010 to 2020). Stanford HIVdb v9.4 was used to interpret resistance profiles. We defined resistance as both intermediate resistance (IR) and HLR interpretations. A sample size of 125 subjects was calculated to allow estimation of the primary endpoint with sufficient precision (95%CI). A cross-sectional analysis was carried out to identify risk factors associated with HLR to ETR.2=4.5, p=0.034).N=125; 81.6% were male. Median age at VF was 39.5 years (IQR: 34.0-35.0); 82,4% received efavirenz and 17.6% nevirapine. Duration of VF: 245 days (IQR 133-413). Median HIV-1 viral load (VL): 11255 c/mL (3606-45100); median CD4+ cell count: 192 cell/uL (86-333). Seventy-seven samples (61.6%) had ETR resistance-associated mutations (RAMs); 25 (20%) had mutations associated with the highest levels of reduced susceptibility . Most frequent ETR RAMS were: 100I (16%), 190A (16%) and 181C (12%); DOR RAMs: 100I (16%), 188L (8.8%) and 106M (4.8%), and RPV RAMs: 100I (16%), 181C (11.2%) and 138A (7.2%). Prevalence of resistance to ETR was 44.8% , resistance to DOR: 64%  and to RPV: 52% . ETR maintained susceptibility to DOR resistant strains in 27.2% of the cases. We found a statistically significant association between HIV-1 VL >10000 c/mL and the risk of developing HLR to ETR (XIn our cohort, resistance to ETR after VF to a NNRTI-containing regimen was lower than to DOR and RPV. HLR was uncommon and associated with high HIV-1 VL. Almost one third of DOR-resistant strains remained susceptible to ETR.Verónica Mingrone, GSK/ViiV: Educational Courses|Janssen Pharmaceutical Companies: Educational Courses Eliana Loiza, n/a, GSK/ViiV: Educational Courses|Janssen Pharmaceutical Companies: Educational Courses Norma Porteiro, n/a, GSK/ViiV: Advisor/Consultant|GSK/ViiV: Grant/Research Support|GSK/ViiV: Educational Courses|Janssen Pharmaceutical Companies: Advisor/Consultant|Janssen Pharmaceutical Companies: Grant/Research Support|Janssen Pharmaceutical Companies: Educational Courses Ezequiel Córdova, n/a, GSK/ViiV: Advisor/Consultant|GSK/ViiV: Grant/Research Support|GSK/ViiV: Educational Courses|Janssen Pharmaceutical Companies: Advisor/Consultant|Janssen Pharmaceutical Companies: Grant/Research Support|Janssen Pharmaceutical Companies: Educational Courses


These results should be taken into consideration for the elderly patients under treatment.COVID-19 inactivated vaccine-induced humoral responses in patients with lung cancer (LCs) to SARS-CoV-2 wild-type (WT) strain and variants BA.4/5 after the primary 2-dose and booster vaccination remained unknown. We conducted a cross-sectional study in 260 LCs, 140 healthy controls (HC) and additional 40 LCs with serial samples by detecting total antibodies, IgG anti-RBD and neutralizing antibodies (NAb) toward WT and BA.4/5. SARS-CoV-2-specific antibody responses were augmented by the booster dose of inactivated vaccines in LCs, whereas they were lower than that in HCs. Enhanced humoral responses waned over time after triple injection, notably in NAb against WT and BA.4/5. The NAb against BA.4/5 was much lower than WT. Age ≥ 65 was risk factor for immunization of NAb to WT. Undergoing treatment resulted in a lower antibody response than those without and radiotherapy was a also risk factor for seroconversion of NAb to WT. Lower lymphocyte counts contributed to a lower titer of IgG anti-RBD and NAb against BA.4/5 in LCs than HCs. Specifically, total B cells, CD4The online version contains supplementary material available at 10.1186/s13045-023-01443-3. Since SARS-CoV-2 spread all over the world, patients with lung cancer (LCs) had an estimated case fatality rate of more than 30%, compared with 0.7% to 8.0% in general population . As the In addition, the Omicron variant harboring 30–40 mutations in the viral spike protein produced high immune evasion . HomologP < 0.05) than the second in LCs. A durability of total antibodies was found   in LCs, but it decreased faster than HCs and the result showed it was 3.8-fold higher in HCs than LCs after 180 days post-booster vaccine (P = 0.0003)  in LCs   in LCs   and Omicron BA.4/5 in LCs (P = 0.0339). The lower immunization may be explained by lower number total B cells, CD4+T cells and CD8+T counts in LCs as their correlation to the humoral response (Additional file Moreover, our results suggested undergoing various anticancer therapies influenced the antibody response of post-booster vaccine. LCs received radiotherapy generated lower level of total antibodies than other therapeutic strategies post-booster inactivated vaccines. The IgG anti-RBD antibodies titer, NAbs against WT and Omicron BA.4/5 were significantly lower in patients receiving various therapies than those without (Fig. Overall, our study revealed strengthened humoral responses post-booster vaccine among LCs, albeit lower than HCs. However, the booster dose failed to establish a potent and durable antibody response for Omicron BA.4/5, which gives rise to the risk of breakthrough infections of Omicron variants, especially in those old and undergoing anticancer therapies. Given the lower antibodies in LCs receiving various active anticancer therapies, further studies are needed to determine whether increased dosage, mixing vaccine types or additional doses enhance immunogenicity.Additional file 1: Methods.Additional file 2: Table S1. Matched demographics between 260 patients with LC and 140 HCs. Table S2. Antibody Response to SARS-CoV-2 inactivated Vaccine between 260 patients with LC and 140 HCs. Table S3. Demographics and clinical characterization of 40 patients with LC with sequential samples. Table S4. Comparative analysis of neutralizing effect responses to SARS-CoV-2 WT and Omicron variant BA.4/5 in 260 patients with LC. Table S5. Antibody response to SARS-CoV-2 inactivated vaccination in 260 LCs and 140 HCs aged < 65 and ≥ 65 years. Table S6. Antibody response to SARS-CoV-2 inactivated vaccination in 260 LCs receiving various treatment regimens. Table S7. Risk factors associated with seropositivity of SARS-CoV-2 antibodies in 144 LCs received booster vaccine.Additional file 3: Figure S1. SARS-CoV-2 antibodies response in 40 LC patients after the second or booster dose of inactivated vaccine. Total antibodies against SARS-CoV-2. Concentrations of IgG anti-RBD antibodies. Inhibition rates of NAb against SARS-CoV-2 WT. Inhibition rates of NAb against Omicron BA.4/5.Additional file 4: Figure S2. Comparison of neutralizing effect responses to SARS-CoV-2 WT and Omicron variant BA.4/5 in LCs. The figures show the median and quartiles. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. LC, lung cancer; WT, wild type.Additional file 5: Figure S3. Comparison of lymphocytes counts in 260 LCs and HCs. The figures show the median and quartiles. **P < 0.01.Additional file 6: Figure S4. Correlation of biological variables and magnitude of SARS-CoV-2 antibodies after the booster dose.


The aim of this prospective multicentre, French observational study was to describe the conditions of Ceftolozane/Tazobactam (C/T) use in hospital settings, and outcomes. This sub-analysis focuses on the cystic fibrosis (CF) patients included in the overall cohort.Adult patients suffering from CF having received at least one dose of C/T and followed up as per routine clinical practice until stop of C/T were included in this analysis. Additional data related to CF were collected.Between October 2018 and December 2019, 63 patients with CF were enrolled from 28 sites. Mean age was 33.6 years and 44.4% were males. 12 patients (19.0%) received a lung transplant, 46.0% had comorbidities with diabetes (34.9%) being the most frequent. Most patients (69.8%) had normal renal function, almost one-third (28.6%) were immunosuppressed. One-half of patients presented with class 2 mutation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. None of the patients received CFTR modulators due to non-availability during the study period.About one-half of patients (52.4%) experienced intolerance/allergy to antibiotics; of these, 78.1% was associated with Ceftazidime.Pseudomonas aeruginosa was the most predominant pathogen representing 89.9% (71/79) of the isolates. C/T showed a very high susceptibility rate of 91.5% across those strains (Table 1). C/T demonstrated higher susceptibility rates than other B-lactam agents (Table 2).Microbiology results showed that Treatment duration until complete cure had a median of 15 days and occurred in 44.4% of patients. Treatment duration until partial cure or end of treatment was a median of 15 days and occurred in 46% of patients (Table 3). Only 2 patients experienced an adverse event leading to a discontinuation of treatment.Treatment with C/T had a positive impact on pulmonary function measured by FEV1 (Table 4) with a mean increase after treatment from 1.33L (range 0.5 - 4.1L) to 1.47L (range 0.6 - 4.6L).Pseudomonas aeruginosa.These results suggest that C/T is an effective and safe option for the treatment of CF patients with a potential positive impact on FEV1 when treating bacterial infections mainly caused by Xavier Bourge, PharmD, Merck: Employe Brune Akrich, MD, MSD: Employee David Boutoille, MSD France: Board Member Isabelle Brassac, Scientist, MSD France: salary Carole Mackosso, n/a, MSD France: MSD employee Jean-François Timsit, MD, merck: Advisor/Consultant Bernard Castan, MD, ADVANZ: SPEAKER MODERATOR|BIOMERIEUX: SPEAKER MODERATOR|GILEAD: Advisor/Consultant|MSD France: Board Member|MSD France: SPEAKER MODERATOR|SANOFI: Advisor/Consultant|SHIONOGI: Advisor/Consultant Pierre-Regis Burgel, MD,PHD, ASTRA-ZENECA: Honoraria|BOEHRINGER INGELHEIM: Honoraria|CHIESI: Honoraria|GSK: Honoraria|INSMED: Honoraria|NOVARTIS: Honoraria|PFIZER: Honoraria|VERTEX: Grant/Research Support|ZAMBON: Honoraria


Burkholderia cepacia complex (Bcc) and Burkholderia gladioli strains. The addition of taniborbactam to cefepime shifted cefepime minimum inhibitory concentrations toward the provisionally susceptible range in 59% of the isolates tested. Therefore, cefepime-taniborbactam possessed similar activity as first-line agents, ceftazidime and trimethoprim-sulfamethoxazole, supporting further development.The novel clinical-stage β-lactam-β-lactamase inhibitor combination, cefepime-taniborbactam, demonstrates promising activity toward many Gram-negative bacteria producing class A, B, C, and/or D β-lactamases. We tested this combination against a panel of 150  Pseudomonas aeruginosa, including strains producing VIM-2  producers (Taniborbactam (formerly VNRX-5133) is a novel bicyclic boronic-acid β-lactamase inhibitor  being deng VIM-2 –11, 13roducers , 14–17Burkholderia cepacia complex (Bcc) is a group of >20 related non-fermenting pathogens (Burkholderia gladioli can cause chronic infections in the immunocompromised and those with cystic fibrosis (CF) with 8%–10% of CF patients acquiring Bcc or B. gladioli by end stage, leading to increased morbidity and mortality  and AmpC (class C) β-lactamases  as well as β-lactam-β-lactamase inhibitor combinations   , while tic acid) . Severalin vitro , 28–30isolates , 28, 29.ambifaria, arboris, cenocepacia, cepacia, contaminans, diffusa, dolosa, multivorans, pseudomultivorans, pyrrocinia, seminalis, stabilis, ubonensis, and vietnamiensis) and 10 B. gladioli from the Burkholderia cepacia Research Laboratory and Repository (University of Michigan) . Minimum inhibitory concentration (MIC) results were interpreted using Clinical Laboratory Standards Institute (CLSI) breakpoints, where available (P. aeruginosa of susceptible (≤8 µg/mL), intermediate (16 µg/mL), and resistant (>32 µg/mL)   , 25, 31 vailable . Cefepimsistant > µg/mL (3nd lung) –17 andnd lung) , 34. Theli panel , 32. Of 50 value decreased by fourfold from 32 to 8 µg/mL  shifted the MICs toward the provisionally susceptible range; the MICge panel . MoreoveblaPenA , 31. Poiocepacia . Alteratdomallei ; efflux  species .Burkholderia species with MIC50 of 4 µg/mL compared to 32 µg/mL for cefepime. The addition of 8 µg/mL of vaborbactam further increased the potency of meropenem with meropenem-vaborbactam yielding an MIC50 of 2 µg/mL. Levofloxacin performed similarly to cefepime alone with 26% vs 23% of isolates testing susceptible, respectively. Notably, only 4/150 strains (3%) possessed high MIC values to cefepime, cefepime-taniborbactam, meropenem, meropenem-vaborbactam, and levofloxacin. The rank order of potency by percent susceptibility or percent provisional susceptibility (≤16 µg/mL for cefepime-taniborbactam) against these Burkholderia spp. was as follows: meropenem-vaborbactam (89%S) > cefepime-taniborbactam (69%S) > meropenem (55%S) > levofloxacin (26%S) > cefepime (23%S)  but much lower than relebactam and vaborbactam 3.2 and 38 µM, respectively (koff rate was for taniborbactam. These results demonstrate that the efficacy of cefepime-taniborbactam is largely the result of the taniborbactam inhibition of PenA1 β-lactamase. Taniborbactam and avibactam inhibited PenA1 in vitro with relatively similar potencies, while relebactam and vaborbactam exhibited weaker inhibitory kinetic profiles in comparison. In summary, cefepime-taniborbactam will be a welcome addition to the antibiotic arsenal due to its broad-spectrum activity against Enterobacterales and P. aeruginosa producing class A, B, C, and D β-lactamases. With broader analyses, cefepime-taniborbactam may also be valuable to treat infections caused by Burkholderia species.The parameters for inhibition of the ectively  (22, 29,ectively . Moreove


On September 1, 2022, the Advisory Committee on Immunization Practices recommended a bivalent mRNA COVID-19 booster dose for persons who had completed at least a primary COVID-19 vaccination series ≥2 months earlier. Early data showed high effectiveness of a bivalent booster in preventing COVID-19-associated hospitalization within 45 days of receipt; however, little is known about the durability of this protection.Data from the Investigating Respiratory Viruses in the Acutely Ill (IVY) Network were used to conduct a case-control analysis measuring bivalent vaccine effectiveness (VE) against COVID-19–associated hospitalization over time. During September 8, 2022–April 1, 2023, immunocompetent, hospitalized adults aged ≥65 years with COVID-19-like illness were enrolled at 25 hospitals in 20 U.S. states. COVID-19 case-patients tested positive for SARS-CoV-2 by a nucleic acid or antigen test within 10 days of illness onset, while control-patients tested negative for SARS-CoV-2 during the same interval. Multivariable logistic regression was used to measure absolute and relative bivalent VE adjusted for age, sex, race and ethnicity, admission date, and U.S. Health and Human Services region. Unvaccinated patients and patients who received 2–4 doses of monovalent-only mRNA vaccine were used as the reference group for absolute and relative VE, respectively. Bivalent VE was calculated for 7–89 days and 90–179 days from booster dose receipt to illness onset.A total of 2,787 immunocompetent, hospitalized adults aged ≥65 years were enrolled in the IVY Network during the study period . Absolute VE of a bivalent booster dose against COVID-19-associated hospitalization was 58% (95% CI=42%–70%) after 7–89 days and 27% (95% CI= -7% to 50%) after 90–179 days. Relative VE of a bivalent booster dose was 54% (95% CI=41%–64%) after 7–89 days and 19% (95% CI= -8% to 39%) after 90–179 days .Bivalent mRNA vaccination provided moderate protection against COVID-19-associated hospitalization within 90 days of receipt among adults aged ≥65 years, with waning protection after 90 days. Additional booster doses could improve protection against COVID-19-associated hospitalization among older adults.Adit A. Ginde, MD, MPH, AbbVie: Grant/Research Support|Faron Pharmaceuticals: Grant/Research Support Ithan Peltan, MD, Asahi Kasei Pharma: Institutional support|Regeneron: Institutional support Emily T. Martin, PhD, MPH, Merck: Grant/Research Support Matthew Exline, MD, Abbott Labs: Honoraria|Regeneron: Grant/Research Support Adam S. Lauring, MD, PhD, Roche: Advisor/Consultant|Sanofi: Advisor/Consultant


Our objective was to assess their Kp clinical isolates were selected. Minimum inhibitory concentrations (MICs) were determined by broth microdilution in triplicate. Time-kill analyses were used to test CZA and MVB alone (1 and 4x MIC) and in combination with colistin , fosfomycin , gentamicin , meropenem , and tigecycline  against 1x108 cFu/mL. 24 hour log-kill was calculated as log cFu/mL decrease from time 0.15 KPC-ompK36 WT, IS5, or GD. 27% and 73% were KPC-3 and KPC-2, respectively. 100% were susceptible to CZA and MVB. None were resistant to COL. 3 were non-susceptible to GEN, which were all IS5 (Table 1).5 isolates each had Figure 1). Mean log-kills did not vary for CZA + COL, GEN, or TGC against ompK36 mutants vs WT; however, log-kills were significantly lower for CZA + FOS  or MEM  against ompK36 mutants compared to WT. Synergy rates were reduced from 100% against WT to 80%, 60%, and 40% against mutant isolates for CZA + COL, MEM, and FOS, respectively .In combination with CZA 1x MIC, each of COL, GEN, and MEM were bactericidal against all isolates; mean ± standard error (SE) log kills were -7.39 ± 0.3, -6.58 ± 0.93, and -5.81 ± 0.21, respectively . Mean ± SE log kills were significantly greater against porin mutants compared to WT for MVB + COL , FOS , or TGC . Rates of synergy were also higher against porin mutants vs WT for MVB + FOS or COL .MVB 1x MIC in combination with COL, FOS, GEN, and TGC were bactericidal 67, 27, 80, and 47% of the time, with corresponding log kills of -3.96 ± 1.1, -1.56 ± 0.83, -5.05 ± 0.82, and -2.53 ± 0.28, respectively  and MVB (meropenem-vaborbactam) at 1X MIC, stratified by ompk36 status: wild-type (WT) versus mutant (MUT). Bactericidal is defined as -3 log kill from time 0 at 24 hours and indicated by the dotted horizontal line. COL = colistin; FOS = fosfomycin; GEN = gentamicin; MEM = meropenem; MIC = minimum inhibitory concentration; TGC = tigecycline.Figure 2Proportion of bactericidal, synergistic, and antagonistic outcomes for ompK36 wild-type  versus mutant  KPC-Kp isolates tested against CZA (ceftazidime-avibactam) or MVB (meropenem-vaborbactam) at 1X MIC as single agents and in combination with colistin (COL), gentamicin (GEN), tigecycline (TGC), meropenem (MEM), or fosfomycin (FOS). Bactericidal activity was defined as -3 log kill from time 0 at 24 hours; synergy and antagonism were defined as -2 or +2 log kill at 24 hours compared to most active single agent, respectively.Porin mutations negatively impact CZA synergy with other antibiotics compared to WT, though 24-hour log kills for COL, FOS, GEN, and MEM remained below the bactericidal threshold. Conversely, porin mutations potentiate synergy between MVB and COL, FOS, or TGC.Ryan K. Shields, PharmD, MS, Allergan: Advisor/Consultant|Cidara: Advisor/Consultant|Entasis: Advisor/Consultant|GSK: Advisor/Consultant|Melinta: Advisor/Consultant|Melinta: Grant/Research Support|Menarini: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Roche: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Utility: Advisor/Consultant|Venatorx: Advisor/Consultant|Venatorx: Grant/Research Support


Scientific Reportshttps://doi.org/10.1038/s41598-019-41543-0, published online 21 March 2019Correction to: This Article contains errors. In Figure 6C the OS image for SCR siRNA is incorrectly duplicated as the Figure 8C OS image for CNT. The correct Figure


Kosakonia pseudosacchari RX.G5M8, a putative methylotroph, was isolated from garden soil in Hong Kong. Its complete genome, a single chromosome of 4,953,935 bp (GC content 53.91%), was established through hybrid assembly. Kosakonia, designated in 2013 after reassignment of four species of Enterobacter  was vortexed with 900 µL 0.9% (wt/vol) saline and a 10-µL aliquot used to inoculate 1 mL of a minimal salts medium (MSM)  containihttps://github.com/FelixKrueger/TrimGalore) , giving 2,137,624 read pairs  totaling ~534 Mbp. Long-read libraries, prepared from the same extracted DNA using the Rapid Barcoding Kit SQK-RBK004 (without size selection), were sequenced via Oxford Nanopore’s Spot-ON Flow Cell (vR9.4.1) and MinION sequencer, with MinKNOW v20.06.2 software and basecalling by Guppy v6.1.5 high-accuracy mode. The final long-read data set, trimmed by Filtlong v0.2.1 (https://github.com/rrwick/Filtlong) , totaled 63,710 reads (984 Mbp) with mean length 15,448 bp . Default parameters were used for all software unless otherwise specified.A paired-end short-read sequencing library (NEB Next Ultra DNA Library Prep Kit) was sequenced via the NovaSeq 6000 platform using an SP PE250 flowcell and v1.5 Reagent Kit. Reads were quality filtered and trimmed using TrimGalore! v0.6.7 (Kosakonia pseudosacchari strain BDA62-3 (CP063425), with an average nucleotide identity of 98.67%.Unicycler v0.4.3  combinedRX.G5M8 possesses a glutathione-dependent formaldehyde detoxification pathway for methanol metabolism . The nit


The primary objective of this study was to characterize meropenem (MERO) pharmacokinetics (PK) in critically ill infants and children with septic shock to develop dosing recommendations to achieve therapeutic drug exposure in this complex population with a spectrum of renal function from augmented renal clearance (ARC) to acute kidney injury (AKI).Children ≥4 weeks old hospitalized with septic shock requiring fluid resuscitation and pressors, receiving MERO 20 mg/kg IV every 8 hr as standard-of-care, were enrolled and studied prospectively from 2019 to 2023. Population PK modeling was used to derive Bayesian post-hoc PK parameters (NONMEM 7.3). Renal biomarkers  were evaluated as covariates. Estimated glomerular filtration rate (e-GFR) was calculated by the modified Schwartz equation.A total of 304 MERO serum concentrations were included from 26 participants. Median age was 12.6 yr (range 1.2-19.6); weight 38.4 kg (10-98); SCr 0.36 mg/dL (0.09-2.57); CYS 448.9 mg/d (178.3-1824.1), and NGAL 176.05 ng/mL (29.3-1000). Median MERO serum concentration was 11 mcg/mL . A two-compartment model with allometrically scaled weight on clearance (0.75) best described the data. Significant covariates  were e-GFR, CYS, NGAL, age, diagnosis of appendicitis and albumin. Using the final model, the median CL was 0.15  L/hr/kg and V1 0.20  L/kg, with GFR 139  mL/min/1.73 m2 . The distribution of MERO concentrations, CL and GFR across quartiles documents wide variations in renal function observed in septic shock, from AKI to ARC .Meropenem Serum Concentration, Meropenem Renal Clearance, and Glomerular Filtration Rate, by QuartilesWide variation in MERO plasma exposure was documented in children with septic shock across the spectrum of renal function. GFR-based dosing recommendations may optimize MERO dosing and improve outcomes in this population.This work was supported by NICHD grant 1 R01 HD095547-01Edmund Capparelli, PharmD, Melinta: Advisor/Consultant