Anaplastic lymphoma kinase tyrosine kinase inhibitors are standard therapeutic agents prescribed for anaplastic lymphoma kinase-positive non-small cell lung cancer, and treatment with these agents has been shown to contribute to long-term survival in patients. However, there is no consensus regarding the course of treatment after the onset of anaplastic lymphoma kinase tyrosine kinase inhibitors related drug-induced interstitial lung disease.Here, we present a case of successful lorlatinib treatment after the onset of drug-induced interstitial lung disease caused by alectinib.A 57-year-old Japanese man was diagnosed with stage IVB non-small cell lung cancer by bronchoscopy, but gene mutation testing could not be performed because of the small amount of specimen. After diagnosis, first-line therapy with cisplatin/pemetrexed was initiated, but the patient developed renal dysfunction. Bronchoscopy was performed again to guide further treatment, and the non-small cell lung cancer was found to be anaplastic lymphoma kinase positive. Alectinib was started after the onset of progressive disease, but it resulted in drug-induced interstitial lung disease, necessitating alternative treatments. He subsequently received nanoparticle albumin bound paclitaxel, which was halted in view of the renal dysfunction. Thereafter, lorlatinib was administered, which was continued without drug-induced interstitial lung disease relapse.Since alectinib can occasionally cause drug-induced interstitial lung disease, as in the present case, lorlatinib may be an option to continue treatment in patients without other treatment alternatives. With the identification of gene abnormalities in anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC), effective ALK tyrosine kinase inhibitors (ALK-TKIs) have been developed for treatment. ALK-TKIs are more effective than cytotoxic chemotherapy, and long-term treatment with TKIs has been shown to prolong the overall survival of patients. Lorlatinib is a novel third-generation ALK TKI that has been shown to be more potent than second-generation TKIs in biochemical and cellular assays and has the broadest coverage of the identified ALK resistance mutations. Additionally, it was designed to cross the blood–brain barrier so as to achieve high exposure levels in the central nervous system (CNS) . BecauseHowever, it is unclear whether lorlatinib can be used after discontinuation of another ALK-TKI owing to adverse events, for example, in cases of drug-induced interstitial lung disease (DILD). Here, we present a rare case showing the feasibility of continuing lorlatinib after the onset of DILD caused by alectinib.18F-fluorodeoxyglucose positron emission tomography/CT (18F-FDG PET/CT) was performed, and abnormal accumulation was observed in the longitudinal hilar lymph nodes , vertebral body, and left pleura. The results of the 18F-FDG PET/CT and other findings led to the diagnosis of lung adenocarcinoma . We recommended rebiopsy considering the possibility of a genetic mutation, but the patient refused the examination because of strong coughing during the first bronchoscopy and progressive symptoms of hoarseness. The patient strongly desired an early treatment, so we initiated first-line therapy with cisplatin (75 mg/m2) and pemetrexed (500 mg/m2) 3 weeks after the first visit. However, the patient developed grade 2 renal dysfunction , making it difficult to continue the therapy . In addition, progressive disease was noted  was initiated after treatment with steroids. However, 3 months later, obstructive pneumonia associated with an increase in the size of the primary lesion was observed . Because of this complication and upon the patient’s request for oral medication, lorlatinib (100 mg/day) was administered as fourth-line therapy. Treatment with antibiotics and lorlatinib reduced the lesion’s shadow, and no recurrence of DILD was observed. Thirty-three months after the start of treatment, stable disease has been maintained, and lorlatinib has been continued  scan showed a tumor, with its major axis measuring approximately 40 mm in the left upper lobe of the lung. The hilar and mediastinal lymph nodes were also swollen Fig. a. PrimarIn the search for advanced lung cancer treatment, driver genes have been identified, and several molecular-targeted drugs have been developed and reported to exhibit remarkable antitumor effects. For ALK-positive NSCLC, alectinib was more effective than crizotinib in phase III trials (ALEX study) and is, therefore, widely used for first-line treatment . LorlatiHowever, a molecular-targeted drug with a clear antitumor effect may still have to be discontinued upon the occurrence of side effects. The overall frequency of DILD associated with ALK-TKIs is 2.14% . AlthougAs of August 2022, no previous study has reported serious lung disease induced by lorlatinib, and it can be administered in normal doses even to patients with renal dysfunction . As seenIn summary, in patients with driver mutation-positive NSCLC, continuation of treatment with TKIs to the maximum extent possible is essential to improve the prognosis. Even after TKI-induced DILD, another TKI may be considered as a treatment option.


Primary intracranial neuroendocrine tumors are extremely rare malignancies with very few cases reported in the world literature. We describe a primary neuroendocrine carcinoma arising from the right cerebellopontine angle, the second case that has been described in this location. The possible origin in this place and treatment are described.A 29-year-old male patient, diagnosed with schwannoma of the right cerebellopontine angle, and treated with radiosurgery at another institution, came to our hospital six months later, The patient presented with a history of rapid progression of numbness on the right side of the face, diplopia, dizziness, vomiting, and facial palsy. On examination, the right cranial nerves V, VI, VII, VIII, and IX were affected. The MRI showed tumor growth occupying the right cerebellopontine angle, with compression of the brain stem and cerebellum. A right retromastoid craniectomy removed the tumor partially and the histopathological examination revealed a high-grade neuroendocrine carcinoma.We describe a primary neuroendocrine tumor of the brain that, despite its rarity, must be considered in the differential diagnosis. There are currently no guidelines for the management of these tumors. According to previously reported cases, surgery is the first line of treatment, followed by radiotherapy or chemotherapy. We consider that such a rare case is needed to be reported for a better understanding of the disease and its neurobiology. Neuroendocrine neoplasms (NENs) are a heterogeneous and uncommon group of malignancies with varied histology. Rarely, these tumors can occur intracranially. There are nine reported cases with a primary origin in the central nervous system (CNS) -10. The Because of the wide and even distribution of the endocrine cells, these tumors can arise in almost any site of the body, but the most common origin is in the gastrointestinal tract and bronchi. Brain metastasis has been reported in 1.3-1.4% of the patients , but priA 29-year-old male, presented at another institution, with progressive right facial palsy, vertigo, nausea, and vomiting. A magnetic resonance image (MRI) demonstrated a right tumor in the internal auditory canal, protruding to the cisterna of the CPA. It was assumed to be a schwannoma of the right vestibulocochlear nerve Figures -1B. The A head MRI showed tumor growth, occupying the right CPA, with compression of the brain stem and extension to the cerebellomedullary cistern Figures -1D. The Histopathological examination revealed a poorly differentiated carcinoma with solid sheets of large, polyhedral cells with amphophilic and ample cytoplasm Figure , with foA thorough evaluation to exclude any evidence of extracranial NENs was performed, including computed tomography (CT) of the thorax, abdomen, and pelvis, which was negative for other tumors. A positron emission tomography-computed tomography (PET-CT) with 18-fluorodeoxyglucose (FDG) was performed to exclude tumor spread that was negative for extracranial NENs. The patient underwent four cycles of chemotherapy (ChT) of cisplatin and etoposide. And trying to strengthen the ChT, a course of monoclonal antibody and an immune-checkpoint inhibitor pembrolizumab was included. Despite this, he experienced worsening of his facial symptoms and numbness of the other side of the face, as well as difficulty swallowing. An MRI eight weeks after surgery revealed huge disease progression and a severe displacement of the brain stem Figures -1H. ThirNeuroendocrine tumors (NETs) are relatively uncommon tumors arising from enterochromaffin cells located in many organs such as the gut and lung . They arThese cells are found diffusely throughout different organs and are well described throughout the gastrointestinal tract, the respiratory tract, and the central nervous system (CNS) -17. ManyIntracranial involvement by a NEN is most seen as hematogenous metastasis. Its presentation is common 13 months after the initial diagnosis, on average. The incidence of patients with NENs having brain metastases is <5%. Furthermore, only 1.4% of metastatic brain tumors are NENs, and most of these lesions originate from the lung. There is no gender preference for the occurrence of brain metastases. When patients do present with metastatic NENs to the brain, they typically present with other local and distant metastases . Brain mPrimary intracranial NECs are rarely mentioned in the literature, accounting for only 11 cases until the preparation of this manuscript (Table One feature that stands out is that 6 out of 11 primary NENs arise extra-axially. Two cases originated from the pineal parenchyma ,8 and thPrimary brain NENs are rare lesions, particularly at the cerebellopontine angle. The first line of treatment for this type of tumor is surgery followed by radiation and chemotherapy. The prognosis of these tumors is correlated to their histological grade. The case we presented was a high-grade tumor with a limited prognosis.


Brain-derived neurotrophic factor (BDNF) has a demonstrated role in promoting memory functions and neuronal survival. A common variant in the BDNF gene, the Val66Met polymorphism, has been found to reduce BDNF expression and increase vulnerability to neurocognitive impairments and age-related cognitive declines. Research suggests that BDNF Val66Met may be associated with hypertension, a well-established risk factor for health and brain functioning across the lifespan. While BDNF is most studied for its effect on learning and memory, its role in moderating other cognitive domains is not as well understood, especially those most affected by hypertension. Furthermore, no study to date has investigated these relationships exclusively with older adults and those at increased risk for cognitive decline. Therefore, the aim of this study was to investigate the effect of BDNF Val66Met and hypertension on the executive function ability of older adults (mean age 71.3±9.2 years) diagnosed with amnestic Mild Cognitive Impairment (aMCI) (N = 108). Results showed that BDNF Val66Met moderated the relationship between hypertension and executive functioning, such that hypertensive carriers of the BDNF Met allele performed significantly worse compared to Val-Val homozygotes, and independent to the effects of aging. These results indicate that genetic and vascular risk interactions can predispose older adults with aMCI to impairments in multiple cognitive domains, and potentially increase susceptibility to further declines or conversion to dementia. These findings have implications for future research on dementia disease pathology, and highlight the importance of strategies that target fixed and modifiable risk factors to promote cognitive resilience.


The authors regret that the names of two co-authors (Jianwei Lu and Li Guo) were spelled incorrectly in the original article. The correct author names are given here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


The authors regret that Paulo Ribeirinha’s name was spelled incorrectly in the original article. The correct spelling of all author names is presented above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


The authors regret that their names were displayed incorrectly in the original article. The correct author names are as presented above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


Clostridioidesdifficile associated disease. Its precise mechanismof action and the origin of limited activity against Gram-negativepathogens are relatively unknown. Herein, treatment with MGB-BP-3alone significantly inhibited the bacterial growth of the Gram-positive,but not Gram-negative, bacteria as expected. Synergy assays revealedthat inefficient intracellular accumulation, through both permeationand efflux, is the likely reason for lack of Gram-negative activity.MGB-BP-3 has strong interactions with its intracellular target, DNA,in both Gram-negative and Gram-positive bacteria, revealed throughultraviolet–visible (UV–vis) thermal melting and fluorescenceintercalator displacement assays. MGB-BP-3 was confirmed to bind todsDNA as a dimer using nano-electrospray ionization mass spectrometryand nuclear magnetic resonance (NMR) spectroscopy. Type II bacterialtopoisomerase inhibition assays revealed that MGB-BP-3 was able tointerfere with the supercoiling action of gyrase and the relaxationand decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichiacoli. However, no evidence of stabilization of thecleavage complexes was observed, such as for fluoroquinolones, confirmedby a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlightadditional mechanisms of action of MGB-BP-3, including interferenceof the action of type II bacterial topoisomerases. While MGB-BP-3′slack of Gram-negative activity was confirmed, and an understandingof this presented, the recognition that MGB-BP-3 can target DNA ofGram-negative organisms will enable further iterations of design toachieve a Gram-negative active S-MGB.MGB-BP-3is a potential first-in-class antibiotic, a StrathclydeMinor Groove Binder (S-MGB), that has successfully completed PhaseIIa clinical trials for the treatment of  MGB-BP-3 also has potent (<1μg/mL) antibacterial activity against methicillin-resistantand methicillin-susceptible Staphylococcus spp., Streptococcus spp., and vancomycin-resistant and vancomycin-susceptible Enterococcus spp.13 There islimited information in the literature regarding MGB-BP-3′sactivity against Gram-negative organisms, possibly reflecting thewell-known challenges of penetration through the double membrane system.Strathclyde MGBs (S-MGBs), based upon a distamycin template,haveshown remarkable anti-infective properties. Their favorable cytotoxicityprofiles to mammalian cells give them selectivity indices that makethem suitable for development as novel drugs. This has enabled extensive MGB-BP-3 1 has sucStaphylococcusaureus show a steep decrease in bacterial viabilityindicative of catastrophic failure of the biochemical machinery withinthe bacterium, rather than a sigmoidal dose-response curve indicativeof interaction with a single molecular target.13 This is perhaps unsurprising as MGB-BP-3, and S-MGBs moregenerally, are thought to bind to many AT-rich sequences within theminor groove of dsDNA, similar to the template natural product distamycin.MGB-BP-3 is hypothesized to bind as a dimer, with two MGB-BP-3 moleculeswithin each binding site, again, similar to distamycin. This bindingmode has been shown for some early S-MGBs, and also more therapeuticallyinteresting alkene-containing S-MGBs but not MGB-BP-3 itself.14 However, there have been limited studies thatspecifically characterize the interaction of MGB-BP-3 with dsDNA.The biological consequences of MGB-BP-3 interacting with dsDNA haverecently been explored using RNA-Seq and DNase I and potassium permanganatefootprinting. It was demonstrated that MGB-BP-3 binds to and inhibitstranscription from multiple essential promoters on the S. aureus chromosome, and that resistant mutantswere unable to be generated by serial passage experiments.13 However, given the molecule’s abilityto bind to many different sites on the bacterial genome, MGB-BP-3has the potential to interfere with other biological processes involvingDNA, although these have not yet been investigated.Dose-response curves of MGB-BP-3againstHerein,an investigation into the origin of MGB-BP-3′s selectiveactivity against Gram-positive bacteria is presented, including synergystudies and fluorescence microscopy. Furthermore, we expand the evidencebase for MGB-BP-3′s multiple mechanisms of action by providinga detailed account of its interaction with dsDNA and interferenceof the action of type II bacterial topoisomerases. Studies also explorereasons for the lack of significant Gram-negative activity to determinewhether there are prospects of developing S-MGBs for these species.S. aureus and Enterococcusfaecalis) and four Gram-negative  isolates (80 0.2 μM) but no measurable MICs (up to 100 μM) againstthe Gram-negative bacteria. Potent activity against Gram-positivepathogens was further demonstrated using an expanded panel of Gram-positivestrains .The selective activity of MGB-BP-3 for only Gram-positive bacteriawas confirmed using a panel of ESKAPE pathogens comprising two Gram-positive to more than 2000-fold (E. coli) compared with MGB-BP-3 alone. This synergismwas confirmed by carrying out checkerboard assays using the same setof Gram-negative bacteria. Fractional inhibitory concentration indices(FICIs) were indicative of strong synergy between MGB-BP-3 and allGram-negative bacteria ,was investigated 1. A highbacteria 1. Checkeynergism 2.Table S2), was used in fluorescence microscopy studies ofthe same panel of ESKAPE pathogens .These results are indicative of the intrinsic resistanceof Gram-negativebacteria to MGB-BP-3 being due to poor intracellular accumulation.To further demonstrate this, a fluorescent analogue of MGB-BP-3, S-MGB-245,which has a similar lack of Gram-negative activity  family pumps (reflecting published PAβN specificity) normembrane permeation is sufficient in isolation to potentiate activityin these strains. There could also be other factors that contributeto elevated resistance.18 Activity was potentiated in strainsknown to be more permeable, K. pneumoniae M6 (PMBN and PAβN) and PAO1 (PMBN only). The latter againsuggests that inhibition of RND efflux pumps in P.aeruginosa is not enough to potentiate activity.Poor intracellular accumulation of MGB-BP-3 is likely to be causedby either efficient efflux, poor membrane permeation, or a combinationthereof. To investigate these possibilities, we carried out potentiationstudies on a range of well-characterized Gram-negative bacteria usingthe membrane permeabilizer, polymyxin B nonapeptide (PMBN), and theefflux pump inhibitor PAβN used in the presence of Mg to minimizeits membrane permeation effects. or PAβN 2. For A.19 Although the effects are likely to be pleiotropic, a tolC mutant showed a significant reduction in the MIC suggestingthat efflux through AcrAB-TolC, or other membrane transporters thatutilize TolC, is a component of reduced efficacy in Gram-negativebacteria  complex, resulted in significantly higher MICsfor MGB-BP-3 but not for ciprofloxacin, suggesting that the observedreduction in susceptibility is perhaps linked to specific featuresof the β-barrel protein complement of the outer membrane , suggesting that the membraneitself remains intact in this directed insertion mutant under theconditions tested.The role of efflux in mediating resistance was confirmedusingdirected insertion mutants from the Keio collection.bacteria 3.Simila strains 2. Convermembrane 3. The MIS. aureus as a bioassay, given its high degree of susceptibility. The data of salmon, afluorescence intercalator displacement assay using gDNA of the ESKAPEpathogens, a native mass spectrometry experiment using a short AT-richDNA oligomer, and an NMR investigation of MGB-BP-3 binding to thesame short oligomer.Tm = 11 °C, Thermalmelt analysis ofMGB-BP-3 binding to salmon gDNA showed significant stabilization ofthe complex, indicative of strong binding .Nano-electrosprayionization mass spectrometry (nESI-MS) was also used to confirm thatMGB-BP-3 binds to dsDNA. Like the natural product distamycin, S-MGBsare thought to bind to AT-rich sequences as a dimer, i.e., two moleculesof S-MGB bind within each binding site on the minor groove of dsDNA.For these experiments, a self-complementary oligomer with an AT-richbinding site (5′-CGCATATATGCG-3′) was used. nESI-MSof the oligomer alone shows the expected DNA duplex in charge states4- and 5- Panel A, 5, and up1H NMR spectrum was acquired on 5′-d(CGCATATATGCG)-3′. The free DNA showed the expectedappearance of five imino proton signals in the region between δ1H = 12.50 and 13.50 ppm, integrating to 10 proton equivalentsfor 10 protons  in slow chemicalexchange from a potential total of 12 imino proton resonances  (Table S5). The DNA sample was subsequentlytitrated with aliquots of a concentrated solution of MGB-BP-3 initiallyto a 1:1 ligand/DNA duplex molar ratio (Table S6). Additionally, new 1H NMR signals were observed growinginto the NMR spectrum in the region δ1H = 8.50–10.00ppm with each ligand aliquot addition and growing to a maximum bythe end point . At the lowest sampletemperature, this region of the NMR data integrated to 10 proton equivalents,consistent with 10 different imino protons. Additionally, six methylsinglet NMR resonances were observed in the same NMR spectrum in achemical shift window consistent with resonances arising from thyminemethyl groups.Low-intensity, broad-lineshape resonances associatedwiththe DNAimino protons of a ligand/DNA complex are characteristic of looserassociation between the ligand and DNA, the formation of a less well-definedcomplex in terms of fit between the ligand and DNA compared with othercomplexes, suggestive of some conformational exchange, or a combinationof some or all of these characteristics.ach step 6d–i. NotWithout further supporting evidence, two possibleexplanationsmay be speculated. These observations may first be explained by thepresence of a completely asymmetric ligand–DNA complex. Sincetwo equivalents of ligand are bound, this explanation would requireassociation of the individual ligands to be bound to the DNA in quitedifferent locations or with quite different orientations or conformationswith respect to each other. Alternatively, the observations may beexplained by the presence of two different symmetrical ligand/DNAcomplexes, each of which gives rise to five imino proton resonancesrepresenting 10 imino protons for each symmetrical complex.1H, 1H] NOESY NMR data of the cooled (1 °C)2:1 ligand/DNA duplex sample were partially assigned. The DNA proton 1H NMR resonances for the 2:1 ligand/DNA complex indicate twouniquely identifiable sets of linked spatial correlations consistentwith a typical data assignment protocol . Chemical exchange links the two sets of data even at thetemperature used to acquire these NMR data. The data may be interpretedby considering two possible ligand binding models  andcell death.22 Thus, it was important to investigateif this mechanism was relevant to MGB-BP-3.Havingdefinitively demonstrated that MGB-BP-3 interacts stronglywith dsDNA, the ability of this interaction to interfere with criticalDNA processes was investigated. While it has been demonstrated thatMGB-BP-3 can interfere with transcription,S. aureus and E. coli enzymes, further suggestingthat the principal reason for the lack of Gram-negative activity isdue to inefficient intracellular accumulation. The lack of activityin the gyrase and topoisomerase IV cleavage assays suggests that MGB-BP-3does not cause the accumulation of DSBs as is observed in the mechanismof action of fluoroquinolones. This is further confirmed by the limitedeffects (<1.5-fold) of MGB-BP-3 on fluoroquinolone-resistant gyrases(E. coli S83L gyrase and S. aureus S84L gyrase mutants) on the supercoilingassay IC50s.MGB-BP-3 interferedwith the supercoiling action of gyrase and the relaxation and decatenationby topoisomerase IV 6. It is E. coli reporter strains, as used previously and a DNA-damaging agent (mitomycin C (MMC)) were used as positivecontrols for the DNA SOS response, and a compound that interacts withthe 30S subunit of the ribosome (doxycycline) was used as a negativecontrol, all treated at 0.5× MIC. A significant fold-inductionof fluorescence for recA and lexA was observed for ciprofloxacin and MMC, while no significant inductionwas seen for doxycycline. MGB-BP-3 did not induce the SOS responsein this assay at 0.5× MIC, with no induction seen for eithergene, comparable to doxycycline, which is known to kill bacteria bya mechanism not linked to DNA damage.23 This experiment confirms that, unlike the fluoroquinolones, whileinterference with the action of type II topoisomerase is likely tobe a contributory part of the mechanism of action of MGB-BP-3, itdoes not induce DSBs and the SOS response.To provide further evidencethat the interference of the actionof type II topoisomerases occurred through a mechanism distinct fromfluoroquinolones, the SOS response was assessed in a series of eviously 8.18 MGB-Table S1), confirming the differentmechanism of action.Further evidence comes from theobservation that MGB-BP-3 is equallyeffective with ciprofloxacin-resistant and ciprofloxacin-susceptiblestrains but also for the new class of drugs as a whole, that the best possibleunderstanding of the mechanism of action is obtained.MGB-BP-3 is a memberof a new class of drugs with a new mechanismof action and satisfies the four WHO criteria for genuine novelty.The literaturedata supporting the development of MGB-BP-3 providestrong evidence for its effectiveness against Gram-positive pathogens;however, there is limited information on its effectiveness againstGram-negative pathogens. In this study, we add to the evidence thatMGB-BP-3 is highly active against Gram-positive bacteria while demonstratingits limited activity against Gram-negative bacteria. As is the casefor many Gram-positive only drugs, MGB-BP-3 appears to be ineffectiveagainst Gram-negative bacteria due to poor intercellular accumulation.This may be due to a combination of poor penetration and efficientefflux, but the specific mechanism appears to vary depending on thecharacteristics of the specific strain. In many cases, strong potentiationwith PAβN and/or PMBN was observed that suggests that MGB-BP-3may be effective against Gram-negative bacteria if an appropriatesynergistic partner can be found, or its structure can be alteredto allow for sufficient accumulation.The full mechanism ofaction of MGB-BP-3 is not well understood,not least because its design adopts a multitargeted approach. Multipleexperimental techniques have confirmed that MGB-BP-3 binds stronglyto dsDNA. Importantly, by confirming that MGB-BP-3 binds to gDNA fromboth Gram-positive and Gram-negative organisms, we have provided furtherevidence that the difference in activity of MGB-BP-3 in these bacteriais due to differential intracellular accumulation, and not a differencein target engagement. The precise interactions of MGB-BP-3 with DNAwere further characterized by demonstrating that the compound bindsas a 2:1 dimer, similar to the natural product distamycin, from whichit is derived. Furthermore, NMR studies strongly suggest the potentialof a dynamic binding interaction with dsDNA, at least for the specificshort, AT-rich dsDNA oligomer used in the experiments.13 Here, MGB-BP-3 has beenshown to interfere with other DNA-centric processes, such as the actionof topoisomerase and gyrase, but it is not thought that MGB-BP-3 interactswith these enzymes directly to achieve this interference. Instead,we suggest that this is likely to be achieved by either MGB-BP-3 directlymasking the enzyme binding site on DNA or indirectly altering thetopology of the binding site through an allosteric mechanism fromproximal binding. Notably, the absence of interference with the cleavageassays and the absence of induction of recA and lexA indicate a different mechanism of action to the fluoroquinolones,which is consistent with MGB-BP-3 binding to DNA and interfering withthe formation of the DNA–enzyme complex.Previousstudies employing RNA-Seq identified ∼700 transcriptswith drug-altered expression profiles, many of which were associatedwith DNA replication, supercoiling, and primosome formation. Moreover,potassium permanganate footprinting confirmed that MGB-BP-3 bindsto certain SigA-dependent promoter regions, preventing transcriptionof these genes.13 Consequently, the evolution of target-basedresistance for MGB-BP-3, and by extension other S-MGBs, should beunlikely.Our study hascontributed to the understanding of the mechanismof action of MGB-BP-3 and has identified additional DNA-centric mechanismsthat MGB-BP-3 can interfere within bacteria. These findings providefurther evidence that the resilience to resistance of MGB-BP-3 seenin the laboratory may be due to its multitargeted design requiringmany simultaneous mutations at the multiple target sites on the genomicDNA.MGB-BP-3 and S-MGB-245were preparedas previously described in refs , respectSalmonDNA  at 1 mg/mL in 1 mM phosphate buffer (pH7.4) containing 0.27 mM KCl and 13.7 mM NaCl was annealed at 90 °C for 10 min. MGB-BP-3 at 10 mM in DMSO wasdiluted with the same phosphate buffer, and combined with the salmonDNA stock to yield a single sample with 10 μM S-MGB and 0.02mg/mL gDNA in 1 mM phosphate buffer containing 0.27 mM KCl and 13.7mM NaCl. Control samples containing only MGB-BP-3 or gDNA were prepared,respectively. Samples were melted at a rate of 0.5 °C/min from45 to 90 °C with spectra recorded at 260 nm on a UV-1900 UV–visspectrophotometer fitted with a Peltier temperature controller (Shimadzhu)using LabSolutions  software. The melting temperatures(Tms) of the MGB-BP-3:DNA complexes were determined by fitting a sigmoidalfunction using a Boltzmann distribution in OriginPro. Two independentexperiments were carried out with values quoted with an error no worsethan ±0.5 °C.Bacterial genomic DNA  or salmon genomic DNA  dissolved in 1mM phosphate buffer pH 7.4  to a concentration of 100 μg/mL inSybrSafe was used as supplied by the manufacturer in DMSO, and MGB-BP-3 wasprepared as 10 mM stock in DMSO. These stock solutions were dilutedappropriately with each other and 1 mM phosphate buffer to give atest solution comprised of 20 μM S-MGB, 12,500-fold dilutionof SybrSafe and 3.92 μg/mL DNA. Control solutions of gDNA andSybrSafe, gDNA, and SybrSafe at these concentrations were also prepared.Test and control solutions were heated to 30 °C and the fluorescenceof each solution was measured using the SYBER filter setting of aStepOnePlus using melt analysis mode (StepOne Software v2.3). Thereduction of fluorescence due to the binding of MGB-BP-3 to the gDNAwas calculated as a normalized percentage based on the fluorescencemeasured due to the control with SybrSafe and gDNA as maximum andthe control with only SybrSafe as minimum. Low normalized percentageindicates a greater ability to displace SybrSafe from the gDNA andsuggests strong binding to gDNA. Three independent experiments werecarried out and the results were presented as average values ±standard error of the mean.DNA oligonucleotidesequence5′-CGCATATATGCG-3′ was purchased in lyophilized form and the purity was confirmed by NMR. Stock solutionsof DNA (100 μM) were prepared with 150 mM ammonium acetate buffersolution  and2 mM potassium chloride solution . This solution was annealed at 90 °C for10 min and allowed to cool to room temperature. S-MGB stocks (10 mM)in 100% DMSO  were diluted to 1 mM S-MGBsolution with 150 mM ammonium acetate. Final samples were preparedfrom this solution to yield final concentrations of 9 μM DNA,100 μM KCl, and 100 μM S-MGB, 1% DMSO. DNA solutions containingno S-MGB included 1% DMSO and were used as controls.via a thin platinum wire . The following instrument parameters were used forthe DNA:MGB-BP-3 complex: capillary voltage 1.2 kV, sample cone voltage80 V, source offset 110 V, source temperature 40 °C, trap collisionenergy 3.0 V, trap gas 4 mL/min. Data were processed using MasslynxV4.2 and OriginPro 2021, and figures were produced using Chemdraw.Nativemass spectrometry (nMS) experiments were carried out on a Synapt G2-Siinstrument  with a nano-electrospray ionizationsource (nESI). Mass calibration was performed by a separate infusionof NaI cluster ions. Solutions were ionized from a thin-walled borosilicateglass capillary  pulled in-house to nESI tip with a Flaming/Brown micropipettepuller . A negative potential inthe range of 1.0–1.2 kV was applied to the solution 2O/D2O atpH = 7.4. A concentrated stock solution of MGB-BP-3 was prepared toallow the addition of 5 μL aliquots of solution up to a maximumtotal addition volume of 50 μL or until a titration end pointwas detected by virtue of no further changes to the NMR data.DNA samples for NMR spectroscopy were preparedas 1 mM duplex solutions solubilized in 550 μL of a 50 mM phosphatebuffer solution prepared with 9:1 H1H NMR data were acquired with excitation sculptingfor solvent suppression (Bruker pulse program zgesgp). The 600 MHzNMR data were acquired using a Bruker AVANCE-II+ NMR spectrometeroperating at 600.13 MHz for 1H resonance on a standardgeometry triple-resonance (TBI-z) probehead equipped for z-pulsedfield gradients with the probe temperature regulated and calibratedfor data acquisition at 298 K. The 800 MHz NMR data were acquiredusing a Bruker AVANCE NEO NMR spectrometer operating at 799.43 MHzfor 1H resonance on a standard geometry 5 mm TCI-z cryoprobeprobehead. Sample temperatures were varied within the range of 298–274K and stabilized prior to data accumulations. In both instances, datawere acquired with 128 transients over a 1H frequency widthequivalent to 20.0276 ppm centered at 4.694 ppm into 32 K (600 MHz)or 64 K (800 MHz) data points using a relaxation delay of 2.0 s betweentransients. For the excitation sculpting routine, sinc soft pulses(bandwidth ∼125 Hz) were used for selective inversion at thesolvent frequency together with smoothed square-shaped gradient pulses(1 ms duration) in a ratio of 31:11. 2D  NOESY NMR data were acquired at 800 MHz on the MGB-BP-3/DNA complexstabilized at 274 K (Bruker pulse program noesyesgpph) into 4 K datapoints for each of 1024 t1 increments over ω1 and ω2 frequency widths equivalent to 20.1756ppm using 64 transients per increment. A mixing time of 250 ms wasapplied to allow development of the nOe and chemical exchange responses.Equivalent reference data were acquired for the ligand-free DNA sampleat 600 MHz by way of creating a reference data set. All raw data wereprocessed within the TopSpin 4.0.5 environment. Fully processed 2DNMR data were imported into the NMRFAM-Sparky environment (version1.414 powered by Sparky 3.13525) for datainterpretation and reduction.1D These experiments were contracted outto Inspiralis Ltd. . In all experiments, the activityof the enzyme was determined prior to the testing of the compoundand 1 U defined as the amount of enzyme required to fully supercoil,decatenate, or relax 0.5 μg of the substrate in 30 min. Cleavageactivity was separately determined and 1 U defined as the amount ofenzyme that gave maximum cleavage without degradation of the substrate.S. aureus and S84L gyrase for E. coli wereused.MGB-BP-3 was tested over a range from 0.01 to 100 μM  or 0.05–500 μM (cleavage)and added to the reaction before the addition of the enzyme. FinalDMSO concentration in the assays was 1% (v/v) for supercoiling, decatenation,and relaxation, and 5% for cleavage. All enzymes and DNA substratesused were obtained from Inspiralis Ltd. . For fluoroquinolone-resistantgyrase supercoiling assays, S83L gyrase for 2, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% (w/v) glycerol,and 0.1 mg/mL BSA. Each reaction was stopped by the addition of 30μL of chloroform/isoamyl alcohol (24:1) and 20 μL of StopDye , 10 mM EDTA, 0.5 μg/mLbromophenol blue) before being loaded on a 1.0% TAE . The gels were run at 90 V for 2 h.DNA gyrase (1 U) was incubated with 0.5 μg of relaxed pBR322DNA in a 30 μL reaction at 37 °C for 30 min under the followingconditions: 35 mM Tris–HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.8 mM spermidine, 6.5% (w/v) glycerol, and 0.1mg/mL BSA. The reaction was then incubated for a further 30 min with2% SDS and 0.5 μg/mL proteinase K. Each reaction was stoppedby the addition of 30 μL of chloroform/isoamyl alcohol (24:1)and 20 μL of Stop Dye , 10 mM EDTA, 0.5 μg/mL bromophenol blue) before being loadedon a 1.0% TAE  gel, runat 90 V for 2 h.DNA gyrase (1 U)was incubated with 0.5 μg of supercoiled pBR322DNA in a 30 μL reaction at 37 °C for 30 min under the followingconditions: 35 mM Tris·HCl (pH 7.5), 24 mM KCl, 4 mM MgClCrithidia fasciculata) in a 30 μL reaction at 37 °C for 30 min under the followingconditions: 50 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate,10 mM magnesium acetate, 10 mM dithiothreitol, 1 mM ATP, and 50 μg/mLBSA. Each reaction was stopped by the addition of 30 μL of chloroform/isoamylalcohol (24:1) and 30 μL of Stop Dye before being loaded ona 1.0% TAE gel, run at 90 V for 2 h.Topo IV (1 U) was incubated with 200 ng kDNA (Kinetoplastcatenated DNA purified from E. coli decatenation assayexcept that the substrate was supercoiled pBR322 DNA.As for Topo IV (1U) was incubated with 0.5 μg of supercoiled pBR322DNA in a 30 μL reaction at 37 °C for 30 min under the followingconditions: 40 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate,10 mM magnesium acetate, 10 mM dithiothreitol, and 50 ug/mL BSA. Thereaction was then incubated for a further 30 min with 2% SDS and 0.5μg/mL proteinase K. Each reaction was stopped by the additionof 30 μL of chloroform/isoamyl alcohol (24:1) and 30 μLof Stop Dye before being loaded on a 1.0% TAE gel, run at 90 V for2 h.E. coli gyrase supercoilingexcept that the assay conditions were 40 mM HEPES-KOH (pH 7.6), 10mM magnesium acetate, 10 mM DTT, 2 mM ATP, 500 mM potassium glutamate,and 0.05 mg/mL BSA. The gels were run at 80 V for 3 h.As for E. coli gyrase cleavage exceptthat the assay conditions were 40 mM HEPES-KOH (pH 7.6), 10 mM magnesiumacetate, 10 mM DTT, 500 mM potassium glutamate, and 0.05 mg/mL BSA.The gels were run at 80 V for 3 h.As for 2, 5 mMDTT, 1.5 mM ATP, 350 mM potassium glutamate, and 0.05 mg/mL BSA. Eachreaction was stopped by the addition of 30 μL of chloroform/isoamylalcohol (24:1) and 30 μL of Stop Dye before being loaded ona 1.0% TAE gel, run at 70 V for 2 h.Topo IV (1 U) was incubated with 200 ng of kDNA ina 30 μL reaction at 37 °C for 30 min under the followingconditions: 50 mM Tris·HCl (7.5), 5 mM MgClE. coli topo IV cleavageexcept that the assay conditions were 50 mM Tris·HCl (7.5), 5mM MgCl2, 5 mM DTT, 350 mM potassium glutamate, and 0.05mg/mL BSA.As for 50 data using the followingequation: Exponential Decay, Single, 2 Parameter f = a* exp(−b*x)Bandswere visualizedby ethidium staining for 10 min, destained for 10 min in water, analyzedby gel documentation equipment  and quantitatedusing Syngene Gene Tools software. Raw gel data (fluorescent bandvolumes) were collected from Syngene, Gene Tools gel analysis softwarewas calculated as a % of the 100% control (fully supercoiled DNA band)and converted to % inhibition. The raw gel data were analyzed usingSigmaPlot Version 13 (2015). The global curve fit nonlinear regressiontool was used to calculate IC5 CFU/mLwere incubated with doubling dilutions of MGB-BP-3, prepared froma 10 mM DMSO stock, in a 96-well plate for 20 h at 37 °C in trypticsoy broth (S1) orMHB (Table S2). Optical density was read and the MICdefined as the lowest concentration of compound to inhibit 80% ofvisible growth.The minimum inhibitoryconcentration (MIC) was defined using a microbroth dilution method.Briefly, bacteria at a concentration of 1 × 104  and a stocksolution of 10 mM vancomycin was prepared in sterile water.The reaction was carried out asper the Minimum Inhibitory Concentration procedure except that theinoculum was supplemented with 1 μg/mL lipopolysaccharide (LPS) of compounds at 25 μg/mL kanamycin for 20 h at 37°C with shaking. MICs for each compound were as follows: MGB-BP-3,3.125 μM; ciprofloxacin, 2 μg/mL; mitomycin C, 64 mg/L;doxycycline, 4 mg/L. GFP fluorescence and optical density were measuredand the fluorescence was normalized to cell density. The fluorescenceof the treated samples was divided by the fluorescence of the untreatedsamples to calculate the fold-induction of the promoter.The 5 CFU/mL in MHB and incubating with 1 μM S-MGB-245, orthis and 50 μM PAβN for 1 h. For those experiments withsodium azide, 1 or 10 mM of this was incubated for 1 h before incubationwith S-MGB-245. Subsequently, a 10 μL aliquot was mounted ona glass slide and fitted with a cover slip.A 10 mM stockof S-MGB-245 in DMSO,10 mM stock of PAβN in DMSO, and 100 mM stock of sodium azidein MHB were also prepared. Samples for microscopy were prepared byobtaining log-phase bacteria at a concentration of 1 × 10Images were captured using a ZeissLSM 880 confocal laser scanning microscope and a 63× objectivebefore processing with associated Zen Blue and Adobe Photoshop software.Detection and acquisition parameters were maintained across each speciessample set.


In the Lifestyle Interventions and Independence for Elders (LIFE) trial, a structured exercise intervention slowed kidney function decline in sedentary older adults. Biomarkers of kidney health could distinguish potential mechanisms for this beneficial effect.Randomized controlled trial.A total of 1,381 sedentary adults aged 70-89 years enrolled in the LIFE trial.Structured, 2-year, moderate-intensity exercise intervention versus health education.Physical activity was measured by step count. Primary outcomes were changes in 14 serum and urine biomarkers of kidney health collected at baseline, year 1, and year 2. We determined the effect of randomization on changes in kidney measures and then evaluated observational associations of achieved activity on each measure.Participants assigned to exercise walked on average 291 more steps per day than participants assigned to health education. The intervention was not significantly associated with changes in biomarkers of kidney health. In observational analyses, persons in the highest versus lowest quartile of activity  had significant improvement in urine albumin , −0.37 to −0.06]), alpha-1-microglobulin (−0.18 mg/L [−0.28 to −0.08]), trefoil factor-3 (−0.24 pg/mL [−0.35 to −0.13]), epidermal growth factor (0.19 pg/mL [0.06-0.32]), uromodulin (0.06 pg/mL [0.00-0.12]), interleukin 18 (−0.09 pg/mL [−0.15 to −0.03]), neutrophil gelatinase-associated lipocalin (−0.16 pg/mL [−0.24 to −0.07]), monocyte chemoattractant protein-1 (−0.25 pg/mL [−0.36 to −0.14]), clusterin (-0.16 pg/mL [−0.30 to −0.02]), serum tumor necrosis factor receptor-1 (−0.25 mg/dL [−0.39 to −0.11]) and tumor necrosis factor receptor-2 (−0.30 mg/dL [−0.44 to −0.16]). In sensitivity analyses, incremental changes in activity were most impactful on urine interleukin 18 and serum tumor necrosis factor-1.The original study was not designed to assess the impact on kidney health. Non-white individuals and patients with advanced chronic kidney disease are underrepresented.Randomization to structured exercise did not improve kidney health at a group level. However, higher exercise was associated with concurrent improvements in biomarkers of glomerular injury, tubular function/repair, tubular injury, generalized inflammation, and tubulointerstitial repair/fibrosis.In the Lifestyle Interventions For Elders (LIFE) study, randomization to an exercise and physical activity intervention improved the slope of estimated glomerular filtration rate over 2 years compared with health education among older adults. In this study, we sought to determine whether there were specific biomarkers of kidney health that were affected by the exercise and physical activity intervention to investigate potential mechanisms for this positive impact on kidney decline. We found that randomization to the intervention did not improve any of the 14 measures of kidney tubule health. However, in observational analyses, higher activity was independently associated with improvements in several domains, especially tubular injury and generalized inflammation. These results help to clarify the impact of physical activity on kidney health. Recent trials indicate that physical activity is beneficial in elderly patients with chronic kidney disease (CKD)Biomarkers of kidney health can be loosely classified as representing glomerular damage, glomerular function, tubular injury, tubular dysfunction, and tubulointerstitial repair/fibrosis.14CysC). Furthermore, those who achieved higher activity levels derived greater benefits to eGFRCysC preservation from the intervention. Crucially, the association of higher activity with slower eGFR decline was independent of changes in blood pressure or body weight, suggesting that physical activity can directly mitigate the decline in glomerular function in older adults.The Lifestyle Interventions and Independence for Elders (LIFE) trial was a multicenter trial designed to test the effect of a combined exercise and activity intervention on major mobility and disability in community-dwelling, sedentary older adults.The present study aimed to explore the following: 1) the effects of randomization to an exercise and activity intervention on domains of kidney health beyond glomerular function and 2) the associations of physical activity as measured by step counts with these domains of kidney health. To achieve these objectives, we used urine and serum specimens that were stored at the baseline, year 1 and year 2 visits of the LIFE trial to measure multiple biomarkers reflecting various domains of kidney health. We hypothesized that both randomization to the exercise intervention and higher step counts during the LIFE trial would be associated with relative improvements in biomarkers reflecting glomerular injury, tubular function and repair, tubular injury, generalized inflammation, and tubulointerstitial repair/fibrosis.The LIFE trial was a phase 3 multicenter, randomized controlled trial of a moderate-intensity physical activity and exercise program compared with a “successful aging” health education program, involving 1,635 sedentary older adults enrolled between February 2010 and December 2013 across 8 centers in the United States.All participants in the LIFE study underwent informed consent before participation. This study was approved by the University of California, San Francisco Institutional Review Board (18-25182).Participants in the LIFE trial underwent block randomization stratified by field center and sex. The intervention arm underwent a combined-activity and functional exercise intervention. The activity component targeted 150 minutes of moderate-intensity exercise per week, primarily composed of walking, and the functional exercise component included strength, flexibility, and balance training. Participants were expected to attend exercise sessions at their field center twice weekly and to conduct home-based activities 3-4 times weekly throughout the trial duration. Exercise sessions were individualized, and participants were expected to progress toward a goal of 30 minutes of daily walking, 10 minutes of lower extremity strength training, 10 minutes of balance training, and large muscle flexibility exercises. Intensity started low and increased over the first weeks of the intervention. Participants were asked to walk at a self-perceived exertion of 13 (“somewhat hard”) on the original Borg scale and perform lower extremity strength exercises at a self-perceived exertion of 15-16 (“hard”). The health education arm of the trial involved weekly workshops over the first 26 weeks followed by monthly workshops. These sessions addressed a variety of health topics relevant to older adults  but did not specifically address physical activity.,Baseline assessments conducted before randomization included self-reported socio-demographic information , medical history , and physical examination (body mass index and systolic blood pressure). Physical activity was assessed using accelerometers (ActiGraph wGT3X-BT) at baseline and at 6, 12, and 24 months of follow-up; on each occasion participants wore the accelerometer on their hip for 7 days. Only data collected at baseline and 12 and 24 months were used for these analyses because they were contiguous with the assessments of biomarkers of kidney health. Details of these measures were described elsewhere.During the trial, serum and urine specimens were collected at the baseline, year 1 and year 2 visits and immediately stored at −80 °C. Urine alpha-1 microglobulin (A1M) and albumin were measured using the Siemens BN II Nephelometer. Urine creatinine level was measured with a commercially available colorimetric kit using the Jaffe reaction from R&D Systems. Urine biomarkers  were measured using multiplex assay kits from Meso Scale Discovery (MSD) according to manufacturer protocols. Serum markers  were also measured using a multiplex plate from Meso Scale Discovery (MSD). For each biomarker, all measures for an individual participant were conducted concurrently to avoid assay drift.t tests, Wilcoxon rank sum tests, and χ2 tests were used to compare normally distributed, nonnormally distributed, and discrete characteristics, respectively, between participants with and without accelerometry or biomarker measures. The associations of the intervention on the outcomes of standardized changes in biomarkers of kidney health were analyzed using the mixed effects models with an unstructured covariance matrix. Urine biomarkers were indexed to 1 g of urine creatinine. We standardized the changes in biomarkers to compare the intervention effects across different outcomes qualitatively. The interpretation of the intervention effect was based on the standardized change in biomarker (unit: SD) when comparing physical activity versus health education. Covariates included baseline biomarker, sex and field center (stratified variables), intervention, clinic visit (year 1 and year 2), and intervention-by-visit interaction. Hypothesis tests for intervention effects at the year 1 and year 2 assessment visits were performed using contrasts of the year 1 and year 2 intervention means. Overall comparisons between intervention groups across follow-up visits were obtained using a contrast to compare average effects across both follow-up visits.Baseline characteristics were summarized by randomization groups using means and standard deviations (SDs) for continuous variables or counts and percentages for discrete variables. Here, We next evaluated associations between measured time-updated step count during follow-up with standardized changes in biomarkers of kidney health. The associations between standardized time-updated step counts with standardized changes in biomarkers of kidney health function were determined using mixed effects models. The covariates in the basic model included baseline biomarker, sex and field center (stratified variables), intervention, clinic visit, and intervention-by-visit interaction. Because these analyses were not comparing randomized groups, we additionally adjusted for potential confounding variables, including age, body mass index, systolic blood pressure, race, education, diabetes, cardiovascular disease, and hypertension. All analyses of urine biomarkers were conducted using the biomarkers indexed for urine creatinine. Step count was also analyzed using quartiles for ease of comparing associations with standardized levels of the biomarkers; high versus low quartiles of activity were compared across the intervention period to ascertain the cumulative effect of activity on biomarkers of kidney health.In our prior study,We next attempted to distinguish associations of incremental changes from baseline in activity, as opposed to cumulative effect of activity over 2 years, with parallel changes in the biomarkers of kidney health across the entire cohort. In these analyses, the baseline step count and the changes in step count from baseline were separate covariates for the outcomes of longitudinal biomarkers of kidney health after adjustment for baseline biomarker, intervention, sex, clinical sites, age, body mass index, time-varying systolic blood pressure, race, education, diabetes, cardiovascular disease, hypertension, visit, and intervention-by-visit interaction. Both baseline step count and outcome measures  are standardized. Change in step count was calculated as standardized follow-up step count minus standardized baseline step count.P value of <0.05 was considered to indicate statistical significance. We did not adjust for multiple comparisons because all of the outcomes are interrelated as manifestations of kidney tubule health; our global hypothesis was that physical activity would be associated with improvements in kidney damage and dysfunction. The statistical software used to conduct these analyses included SAS version 9.4 and R version 3.6.1.For all analyses, a 2-sided CysC at baseline was 54 ± 17 mL/min/1.73 m2   .Table 2EIn contrast, when time-updated step count was evaluated comparing high versus low quartiles of activity  or as a Incremental changes in step count from baseline demonstrated significant associations with improvements in tubular injury (IL-18) and generalized inflammation (TNFR1). The coefficients of all remaining biomarkers except for serum KIM1 were directionally associated with improvements in kidney health. Effect sizes of incremental changes in step count were somewhat smaller than the associations with baseline step count .Table 4AIn this analysis of 1,381 community-dwelling sedentary older adults in the LIFE trial, we found no effect of randomization to a structured physical activity and exercise intervention on biomarkers of kidney health, including measures of glomerular injury, tubular function and repair, tubular injury, generalized inflammation, and tubulointerstitial/fibrosis. However, in observational analyses, those participants who had higher time-updated step counts showed improvements in several domains of kidney health. Specifically, participants who walked more had trajectories reflective of less glomerular injury , better tubular function (lower urine A1M and TFF3), better tubular repair (higher urine EGF and UMOD), less tubular injury (urine IL-18 and NGAL), less generalized inflammation (TNFR1 and TNFR2), and less tubulointerstitial fibrosis/repair (urine MCP1 and clusterin). In sensitivity analyses that distinguished the incremental effect of increased activity from baseline on changes in biomarkers of kidney health, we observed that changes in activity appeared most impactful in the domains of tubular injury (IL-18) and generalized inflammation (TNFR1).CysC (as shown previously),,Our findings are somewhat contradictory as randomization to the physical activity intervention did not result in a significant change in any kidney biomarker except for eGFRThere are relatively few prior studies evaluating the effects of exercise on markers of systemic inflammation and/or glomerular health, and they have reported mixed results. Prior studies have differed from this LIFE ancillary study in several ways: smaller sample sizes, restriction to specific stages or etiologies of CKD, shorter exercise interventions , and a narrower profile of kidney health measures. For example, a study of home-based aerobic exercise and resistance training for participants with stage 4 CKD showed no improvement in urine albumin over 6 months, but did show improvement in urinary liver-type fatty acid-binding protein (a marker of tubular injury) and C-reactive protein .,These results help to elucidate the mechanisms by which activity and exercise might affect changes across multiple dimensions of kidney health. Importantly, in addition to a reduction in glomerular filtration rate, the aging kidney has declining proximalIt is important to note that although step counts are a measure of physical activity, this study was geared toward studying the effect of a combined-activity exercise intervention on changes in biomarkers of kidney health and not activity alone. However, the trial only included 2 measures of observed activity that could be used to evaluate a dose-response relationship between components of the intervention and changes in kidney health: step counts and time spent in moderate-intensity activity. In our prior study,There are several other limitations to be acknowledged. This is an ancillary study to an existing trial, and the original study was not designed to test the impact of physical activity and exercise on kidney health. Non-White individuals were underrepresented, and the study included few participants with advanced CKD. Furthermore, given LIFE’s focus on older individuals, these findings may not apply to a younger population. Due to the timing of specimen collection, our analyses were limited to a 2-year follow-up; we do not know whether any beneficial effects of physical activity would be increased or decreased during longer follow-up periods. There are other possible factors that can influence the ability to maintain activity as well as kidney health that this study is not able to quantify, including socio-economic factors affecting access to nutrition, transportation, or caregiving. However, the findings are both thematically and directionally consistent with the randomized effect of physical activity on improvements in eGFR, and the markers assessed here are used to understand potential mechanisms. Nonetheless, the associations of higher step counts with changes in biomarkers of kidney health are observational and are subject to residual confounding effects despite our adjustment for multiple covariates.In summary, in a trial that employed a structured physical activity and exercise intervention for older adults, no differences in biomarkers of multiple domains of kidney health were observed between individuals randomized to structured physical activity intervention and those randomized to health education alone. However, those participants who had higher step counts had relatively improved biomarkers of kidney health, including glomerular injury, tubular function, tubular injury, generalized inflammation, and tubulointerstitial repair/fibrosis. Incremental changes from baseline in activity appeared most impactful on the domains of tubular injury and generalized inflammation. These results help to clarify the overall impact of physical activity on kidney decline. Future research should explore whether these same biomarkers of kidney health change over time as a natural course of aging and, if so, whether physical activity and exercise can slow the natural process of aging in the kidney. Additional studies are needed to evaluate which components of physical activity are most beneficial for kidney health, whether exercise intensity gives a graded benefit in kidney health, and whether persons with advanced kidney disease also benefit from physical activity.


There are limited longitudinal studies on the effects of the COVID-19 pandemic on mental health and well-being, including the effects of imposed restrictions and lockdowns.This study investigates how living in a pandemic, and related lockdowns and restrictions, affected the mental health of people living in Australia during the first year of the COVID-19 pandemic.A total of 875 people living in Australia participated in a longitudinal survey from 27 May to 14 December 2020. This time period includes dates that span pre-, during and post-wave 2 lockdowns in Australia, with strict and sustained public health measures. Linear mixed models were fitted to investigate the effect of lockdown on depression and anxiety symptoms.Symptoms of depression and anxiety improved over time, during and after lockdowns. More adverse mental health symptoms were observed for people with a history of medical or mental health problems, caring responsibilities, more neurotic personality traits or less conscientiousness, and for people who were younger. People who reported being more conscientious reported better mental health.Despite notoriously strict lockdowns, participants did not experience a deterioration of mental health over time. Results suggest a lack of significant adverse effects of lockdown restrictions on mental health and well-being. Findings highlight cohorts that could benefit from targeted mental health support and interventions, so that public policy can be better equipped to support them, particularly if future strict public health measures such as lockdowns are being considered or implemented for the COVID-19 pandemic and other disasters. These occurred in the context of fear and anxiety about how the COVID-19 virus might affect individuals, communities, healthcare services and society. Although public health restriction measures were necessary to ‘stop the spread’, particularly in the absence of vaccines in the early stages of the pandemic, there have been psychosocial (and economic) costs as a result.2The potential for the COVID-19 pandemic to affect mental health and well-being was recognised early on in the pandemic, as a result of concerns around contracting the virus itself as well as from the measures used to contain its spread.3 Studies have reported increased levels of psychological distress and symptoms of depression and anxiety compared with pre-pandemic levels.8 COVID-19 restrictions have also affected social connectedness, with reports of increased social isolation and loneliness associated with increased psychological distress.9 Other downstream effects of public health measures, including job losses and reduced financial resources, have also adversely affected mental health.11 Furthermore, mandatory contact tracing and quarantine have contributed to feelings of anxiety and guilt about the effects of infection, quarantine and stigma.12 COVID-19 infections and associated restrictions have also shown to contribute to, or exacerbate, existing chronic mental illness, including anxiety, depression, post-traumatic stress disorder and substance misuse.13It is crucial to understand how the virus itself and associated restrictions affected the short- and long-term mental health and well-being of people, and how these factors track over time. For these reasons, an international call to action was made for high-quality data on the mental health effects of the COVID-19 pandemic.8 There are limited longitudinal mental health and well-being studies about people living in Australia during the pandemic, and only one of these reported data collected before the second wave.15 This study reports results of one of the few surveys in Australia that capture data leading into and following the second wave, and the infamously strict restrictions imposed during that time.However, the majority of studies examining the mental health and well-being effects of the COVID-19 pandemic in Australia are based on cross-sectional data collected in the early days of the pandemic, when public health orders were not as strict and threat of community transmission was lower (i.e. the first wave). Those studies, which reported that mental health and well-being was substantively lower than pre-pandemic normative estimates, may have captured the immediate, acute shock of a global pandemic following a severe bushfire season.16 and wave 3 from August 2021 until October 2021 (from the Delta variant of COVID-19).17 In 2020, there were 899 deaths from COVID-19 registered in Australia, and 89% of deaths were in Victoria (n = 800).16In Australia, widespread restrictions of movement, distancing measures and physical isolation, or ‘lockdowns’, were implemented from March 2020 as a response to Australia's first locally transmitted case of COVID-19. Up until October 2021, Australia had three major ‘COVID-19 waves’ or distinct peaks of infections that led to widespread lockdowns. The general dates are: wave 1 in March and April 2020 , wave 2 from end of June to October 2020 (mainly affecting the state of Victoria with most infections acquired via community transmission)18 The government's strict lockdown strategy was effective in eliminating community transmissions,19 and restrictions were eased in Victoria from 26 October 2020, when zero community transmissions were recorded in all Australian states. There was an extended period in Australia with no community transmissions until the third wave began. When the last lockdown in Victoria ended on 21 October 2021, the people of Melbourne (a city of 5 million people) had spent a cumulative 262 days under strict lockdown conditions since March 2020, one of the most stringent in the world, leading to the nickname ‘world's most locked down city’.20 These lockdowns were characterised by long periods of border closures, imposed curfew, childcare and school shutdowns, closure of retail and hospitality, restrictions on movement (≤5 km from home) and restrictions of visitors to homes, with only four essential reasons to leave home . Getting a COVID-19 vaccination was only added as a fifth reason to leave home in April 2021, when the first vaccines were available.Most Australians experienced stay-at-home orders during wave 1 of COVID-19 infections. These first wave restrictions were easing in May 2020 (when our survey began collecting data), with single-digit daily case numbers in Victoria at the start of June 2020. However, case numbers were soon to rise as a result of hotel quarantine breaches, resulting in the second wave. The state of Victoria subsequently experienced a large and prolonged second wave of COVID-19 infections, which resulted in approximately 19 000 recorded cases and 800 deaths, linked primarily to 222 outbreaks in residential aged care homes.22 Our analysis of baseline cross-sectional data from this survey,23 collected from 803 participants during the peak of the second wave (from 27 May to 19 August 2020), revealed that self-rated knowledge about COVID-19 was high, adherence to government recommendations to prevent the spread of COVID-19 was high and trust in the Australian government's handling of the COVID-19 pandemic was high, as were participants’ confidence in the Australian healthcare system during the COVID-19 pandemic. Participants also strongly believed that access to essentials  would be maintained.We conducted a longitudinal survey investigating the impact of the COVID-19 pandemic on the mental health and well-being of people living in Australia during the first year of the pandemic, from 27 May 2020 to 14 December 2020. The survey was modelled on the UCL COVID-19 social study, and had the broad aim of exploring the emotional, mental, health and societal behaviours and experiences of people during the COVID-19 pandemic.In the current study, the primary aim was to investigate the impact of the pandemic and major lockdowns on the mental health and well-being of people residing in Australia across three periods: pre-major lockdown, during major lockdown and post-major lockdown in 2020. As Victoria experienced a prolonged lockdown period in 2020, secondary analyses assessed if changes in depression or anxiety differed by participant location , or according to baseline participant characteristics. We hypothesised that greater depression and anxiety symptoms would be observed during the major lockdown period compared with pre- and post-major lockdown. We also hypothesised that people living in areas with longer and more restrictive lockdowns (i.e. Victorians) would experience poorer mental health.Participant recruitment was conducted by the National Ageing Research Institute, using a convenience sampling approach through a variety of channels, including media, social media, targeted advertising, and personal and professional networks. Participation was open to any person living in Australia during the survey, aged 18 years or above. Because the pandemic had population-wide effects that were occurring in real time, and because of the time-sensitive nature of this research, a convenience sampling approach was used to enable quick collection of data at a crucial time during the pandemic, and to generate a large sample in a short period of time.https://projectredcap.org/), or via hard-copy paper versions, which could be posted to participants with a postage-paid reply envelope. New participants could join the study and complete the baseline assessment at any point during that period. The survey collected sociodemographic information, a basic health profile, occupational and social roles (e.g. key worker or carer status), and information about living situation. The baseline questionnaire required approximately 20 min to complete and weekly follow-up questionnaires took approximately 15 min, where participants shared ongoing information about their mental health, loneliness, stressors, adherence to restrictions, confidence in government management, and exercise and social behaviours. Study participants were also invited to participate in a single qualitative interview, and findings will be reported separately.Data collection began on 27 May 2020 and continued until 14 December 2020 via an online platform .https://doi.org/10.1192/bjo.2023.65). Of those with complete surveys (n = 920), 45 individuals had missing or invalid postcode information (see Supplementary Table 2). Excluding individuals with incomplete surveys and incomplete/invalid postcodes resulted in a sample size of 875 participants eligible for analyses. See A total of 1223 individuals began the online survey, with 1215 (99.3%) providing written consent to be included in the study. Among those that provided consent, 295 (24.3%) had incomplete surveys; these individuals did not provide data on the outcome measures or for several key covariates of interest required for the analyses 25 tool. These tools align closely with diagnostic criteria for major depressive disorder and generalised anxiety disorder, respectively.24 In both tools, items ask how often symptoms were bothering respondents in the past week. PHQ-9 scores range from 0 to 27, with scores of 5, 10, 15 and 20 representing cut-off points for mild, moderate, moderately severe and severe depression, respectively. GAD-7 scores range from 0 to 21, and scores of 5, 10 and 15 represent cut-off points for mild, moderate and severe anxiety, respectively.Self-reported mental health was assessed at all time points, using two outcomes: depressive symptoms measured with the nine-item Patient Health Questionnaire (PHQ-9)26 was completed by participants once, at baseline. This measures five key dimensions of personality: openness, conscientiousness, extroversion, agreeableness and neuroticism. Scores for each of the five personality dimensions were determined from the mean score across the three items associated with each dimension. Higher scores for each personality trait represent greater strength for that trait.The Short 15-item Big Five Inventory (BFI-S)Victoria was the only Australian state/territory that entered a prolonged major lockdown in 2020. Metropolitan Victoria (i.e. Melbourne) entered major lockdown at a different time to the rest of Victoria. Participant responses were classified as pre-, during and post-major lockdown, according to both the date the survey was completed and the participant postcode of residence.The pre-major lockdown period was defined as between 27 May (study initiated) and 1–9 July 2020 (for Melbourne), and between 27 May (study initiated) and 2 August 2020 . The lockdown period was defined as between 1–9 July and 27 October 2020 (for Melbourne), and between 3 August and 27 October 2020 . The post-major lockdown period was defined as between 28 October and 14 December 2020 (study end).There was variation in the metropolitan Victoria lockdown as some hot-spot postcodes were placed in lockdown before others (further details are in Supplementary Table 3). Participants living outside of Victoria were considered as a comparator group. For the comparator group, the pre-major lockdown period was considered to be the period before the whole of Victoria being in major lockdown (i.e. 27 May to 2 August 2020).29 Country of birth was included as an exploratory variable to investigate whether separation from family owing to COVID-19-related travel restrictions affected mental health. Area of residence was included as lockdowns varied according to location.Baseline covariates considered to be potentially associated with mental health outcomes included age (years); gender identification ; country of birth ; caring responsibilities for older relatives or friends, or for people with long-term conditions or disabilities ; history of clinically diagnosed mental health conditions ; currently engaged in paid employment, including casual, part-time, full-time or self-employment ; and area of residence . These covariates were selected based on well-known factors considered to be potentially associated with mental health outcomes in the literature.30 This index ranks areas in Australia according to relative socioeconomic advantage and disadvantage based on information from the 5-yearly Census of Population and Housing. SEIFA quintiles were considered ranging from 1 (most disadvantaged) to 5 (least disadvantaged).Area-level socioeconomic status was derived by linking participant postcodes to the 2016 Australian Bureau of Statistics Socioeconomic Index For Areas (SEIFA) Index of Relative Socio-economic Advantage and Disadvantage (IRSAD).Secondary analysis within the Victorian subsample examined which of the following time-fixed risk factors and personality traits were associated with the change in mental health over time across the three time periods: pre-major lockdown, during major lockdown and post-major lockdown in Victoria: (a) increased risk because of older age ; (b) caring responsibilities (yes/no); (c) history of mental health conditions (yes/no); (d) medical history/comorbidities ; (e) country of birth ; (f) essential/key worker ; (g) having one or more children in the household (yes/no); and (h) personality traits as measured by the BIF-S, i.e. extroversion, agreeableness, openness, conscientiousness and neuroticism.31 Participants were defined as having a medical history of comorbidities if they reported having high blood pressure, diabetes, heart disease, lung disease, cancer or other chronic physical health condition or disability.Participants were considered at increased risk of serious COVID-19 outcomes if they were aged 65 years and above with a medical condition or aged above 70 years, as per the Department of Health's definition.n = 875) were calculated along with missing data proportions , an interaction between area of residence and lockdown period was included in adjusted LMMs. In secondary analyses, an interaction between each potential time-fixed baseline risk factor and lockdown period was included in adjusted LMMs, to assess if these factors were associated with differences in depression or anxiety symptoms over time for Victorian participants. The eight factors listed above were considered in separate LMMs in this exploratory analysis.n = 53), as well as any participants who had missing outcome values at all time points for which they provided data . This resulted in a complete-case sample size of 767 (87.7% of the 875 eligible participants) for the PHQ-9 and 764 (87.4%) for the GAD-7. Descriptive characteristics of the full sample, complete-case sample and omitted participants were considered to determine if characteristics were comparable between those who were and were not included in the analysis (see Supplementary Tables 4 and 5). Descriptive characteristics for the complete-case sample and omitted sample of participants were compared to determine if there were any differences in the characteristics of included and excluded participants (Supplementary Table 4). The two samples were generally comparable, although there was a slightly higher percentage of participants who were female (81% v. 74%), employed (56% v. 48%) or had no caring responsibilities (79% v. 74%) in the complete-case sample compared with the omitted sample of participants.A complete-case analysis was conducted, omitting participants with missing data for any of the covariates (n = 767) and GAD-7 (n = 764), 668 (approximately 87%) provided longitudinal data. Each participant on average provided data for 9.7 (s.d. = 7.4) and 9.8 (s.d. = 7.1) weeks (maximum 25) for the PHQ-9 and GAD-7, respectively.Characteristics of the eligible study sample at baseline are shown in Mean pre-lockdown PHQ-9 score was higher than both the mean scores during and post-lockdown, indicating more depressive symptoms before lockdown  and 3. TThe mean pre-lockdown GAD-7 score was higher than both the mean score during and post-lockdown, indicating more anxiety symptoms before lockdown  and 4. TExamination of the changes in mental health over time by participant location in Australia  and 3 shn = 669; 87%) were living in Victoria and experienced strict lockdown measures in 2020. Descriptive statistics are shown in A large proportion of respondents from the sample , compared with those who had no such history . PHQ-9 sHowever, as in the primary analysis, all groups show that depressive symptoms on average decreased slightly over time . There wSimilar to that found for depressive symptoms, GAD-7 scores on average were higher for (a) younger people  across all time periods compared with those who were older , (b) forAs with depression symptoms, there was no interaction effect for any of the potential moderators considered. For all groups, on average, the level of anxiety decreased, indicating that anxiety symptoms improved modestly in this sample over time . For allThe effect of lockdown on symptoms of depression and anxiety were largely unmodified by personality traits  did not experience poorer mental health as a result of the lockdown. Regardless of where participants lived , results indicated symptoms of depression and anxiety improved over time and during and post-lockdowns. However, the change in PHQ-9 and GAD-7 scores was consistently small, with considerable overlap in the confidence intervals of the mean estimates in all analyses, and likely do not reflect clinically meaningful changes. Finally, we found that the effect of lockdowns on symptoms of depression and anxiety were largely unmodified by personality traits. Irrespective of time period, there was a trend for people who scored highly on neuroticism to report more depressive and anxiety symptoms, and conversely, those who self-reported high conscientiousness reported lower levels of these symptoms.27). Additionally, people with caring responsibilities showed more anxious symptoms compared with those with no caring responsibilities. These results are consistent with other studies; despite greater risks and potential difficulties faced by older people during the pandemic and associated restrictions, this age group has been found to report lower rates of psychological distress and poor mental health and lower scores on mood symptoms scales across multiple COVID-19 studies.32 However, findings from the secondary analyses may reflect characteristics of these particular groups, independent of a pandemic. For example, research conducted before the pandemic indicated that depressive or anxiety symptoms tended to decrease with increasing age.33 Additionally, the negative effects of caring on psychological health have been well established, including on anxiety, depression and burden.29 and an Australian study found that carers with unmet support needs faced a two-fold increase in the odds of psychological distress relative to those with no unmet needs.34Secondary analysis of Victorian participants  indicated that mental health generally improved across all groups, with no change in trajectory based on any of the potential modifying factors. People with a history of medical conditions or clinically diagnosed mental health problems, or younger participants , displayed greater depressive and anxious symptoms on average 36 than the pre-lockdown mean reported for this study , which suggests a general increase in depressive symptoms triggered by the pandemic. However, consistent with our results of improved mental health scores across the three time periods, the UCL COVID-19 social study also found improving mental health over time,22 and findings from China indicated that rates of psychological distress declined in the weeks following the initial outbreak.37 Other findings from Australia also showed that people who were still working early in the pandemic showed improved mental health over time.11 This may reflect some adjustment to a ‘new normal’ for individuals whose lives and livelihoods were not severely affected by the pandemic, or a gradual return to the pre-pandemic baseline after the initial outbreak.Our analysis is one of the few surveys in Australia that capture data leading into and following the second wave and the infamously strict restrictions imposed during that time. There are limited studies published to date with longitudinal data for mental health and well-being on people living in Australia during the pandemic, only one of which reported data collected before the second wave.38 Another longitudinal survey, conducted between April and September 2020, found increased depressive symptoms and poorer self-reported coping, hopefulness and quality of life during the second wave of the pandemic, compared with the first wave.15 Hopefulness, however, rebounded in September 2020 across their sample, likely because of to the impending easing of government-imposed restrictions. Anxiety, stress and resilience, on the other hand, remained relatively stable. Other research has consistently shown that mental health and psychological well-being have been adversely affected during the pandemic by job loss or reduced work, financial stress, pre-existing mental health disorders, feeling that government restrictions were negatively affecting their daily lives or that they would continue for the long term, loss of social connectedness, or worry about contracting COVID-19.27 The analyses presented in this study controlled for as many of those factors as the available data would allow, to examine the effect of pandemic-associated restrictions on mental health. Taken together, the present results and other findings in the literature suggest that individual experiences of the pandemic and resultant effects on mental health are driven by the ways in which public health measures and restrictions have directly affected the lives of people rather than being in lockdown per se. It is, therefore, necessary to examine individual factors that may influence the way in which lockdown may affect participants’ mental health.In contrast, two cross-sectional surveys administered in April 2020 and July to August 2020 found substantially greater prevalence of clinically depressive and generalised anxiety symptoms in Victoria compared with other states and territories during the second wave.39 which would have affected mental health. Caution should therefore be taken in generalising our results to the wider population. Participants could also both join and leave the study at different time points, meaning that participants that contributed data during the pre-lockdown period may differ to participants that contributed data during the lockdown or post-lockdown periods. This means that differences observed could be attributable to differences between participants rather than within-individual improvements in mental health. Thus, improvements in mental health over time may not be a result of within-individual changes in mental health but could be explained by the number of participants who have provided outcome data at the different lockdown stages. However, our sensitivity analyses of participants that provided data across all time periods provided consistent findings (see Supplementary Table 10).Although significant in our model, the point estimates or the limits of the confidence intervals did not reflect clinically meaningful differences for either depression or anxiety symptoms . Although the PHQ-9 and GAD-7 are well-validated scales for the purpose of assessing depression or anxiety symptoms, it is important to note that these measures were collected via self-report and are not diagnostic data, and as brief self-report instruments, are not as valid as more detailed measures. There are also some self-selection biases to consider. Convenience sampling likely resulted in sampling bias. Most participants were women (80%), very few opted for the option of hard-copy surveys and the survey was predominately conducted online, which may have skewed the sample of participants toward people who were more able and willing to access the necessary technology. Additionally, nearly 50% of the sample were in the most advantageous socioeconomic quintile and were in current employment, which skewed our sample as those in more socioeconomically disadvantaged and diverse circumstance experienced disproportionately higher mortality and morbidity associated with the virus,In summary, this study reports a lack of deterioration in mental health over time in people living in Australia during the first year of the COVID-19 pandemic, even throughout notoriously strict lockdowns and restrictions. These results are meaningful as there are limited longitudinal survey studies on the well-being effects of COVID-19, especially those that consider the ways in which public health measures and restrictions directly affect the mental health of people. Our findings reveal the cohorts that appear to be at greater risk of poor mental health and should be targeted for mental health supports and interventions. These were people with a history of clinically diagnosed mental health problems, younger participants , people with a history of medical conditions, people with caring responsibilities and people with more neurotic personality traits. For example, given that high conscientiousness was associated with better mental health in our study, targeted mental health supports could focus on improving conscientiousness in at-risk groups. This also suggests that public health messaging highlighting social responsibility may resonate with, and thus support the mental health of, highly conscientious people during a pandemic; however, further research is required to investigate this. Other future research should examine the short- and long-term effects of job loss, financial stress and social isolation on mental health, to better understand differential effects of lockdown consequences on mental health trajectories across the pandemic.These results provide insight into how people are navigating the pandemic and tracking mental health across time. This may help researchers, clinicians and policy makers to better understand the effects of COVID-19 and restriction measures on individuals, identify which groups are most at risk, and inform the advice that people are given about how to maintain mental well-being and mental health during a pandemic. As the pandemic and its extended effects continue, investigations such as this study into the impact of the COVID-19 pandemic on mental health are vital for informing service delivery and supports and providing more evidence, as future strict public health measures such as lockdowns are considered for pandemics or other public health reasons.