Brief Report: Clinical Outcomes by Infusion Timing of Immune Checkpoint Inhibitors in Patients With Locally Advanced NSCLC.

Introduction: Previous studies reported an association between immune checkpoint inhibitor infusion timing and the treatment effect in metastatic NSCLC. The present study assessed the association between durvalumab infusion timing and survival outcomes in patients with locally advanced NSCLC. Methods: Patients receiving durvalumab after chemoradiotherapy for locally advanced NSCLC at a single institution were retrospectively analyzed, and the association of the proportion of durvalumab infusions greater than or equal to 20% versus less than 20% after 3 PM with progression-free survival (PFS) and overall survival was assessed. Results: A total of 82 patients were included, with a median age of 69 years (interquartile range, 62-74 years); of these, 67 patients (82%) were of male sex, and 78 patients (95%) had a history of smoking. The median number of durvalumab infusions per patient was 16 (interquartile range, 8-24). Patients with at least 20% of their durvalumab infusions after 3 PM (n = 12/82, 15%) had a significantly shorter PFS than those who did not (median: 7.4 mo versus not available [NA]; hazard ratio [HR], 2.43; 95% confidence interval [CI]: 1.11-5.34, p = 0.027), whereas overall survival was shorter among the former compared with the latter group (median: 22.4 versus NA; HR, 1.80; 95% CI: 0.73-4.42, p = 0.20). In addition, both backward stepwise multivariable analysis and propensity score-matching analysis revealed that receiving at least 20% of durvalumab infusions after 3 PM was significantly associated with worse PFS (HR, 2.54; 95% CI: 1.03-5.67, p = 0.047; and HR, 4.64; 95% CI: 1.95-11.04; p < 0.001, respectively). Conclusions: The time of day of durvalumab infusions may impact survival outcomes in patients with locally advanced NSCLC.

A case of paroxysmal cold hemoglobinuria complicated by latent syphilis.

An 80-year-old man presented in December with the main complaint of jaundice. Blood tests revealed hemolytic anemia and renal dysfunction. Positive syphilis serology results led to a diagnosis of untreated latent syphilis. A positive direct Coombs test led to a diagnosis of autoimmune hemolytic anemia (AIHA). Antibiotics were started for the syphilis, with improvement in the anemia and renal dysfunction observed. However, paroxysmal intravascular hemolysis occurred after his discharge. Based on a positive Donath-Landsteiner (D-L) test, paroxysmal cold hemoglobinuria (PCH) diagnosis was made. The hemolytic anemia improved after further treatment for syphilis, and further avoiding exposure to cold.

E3MPH16: An efficient endosomolytic peptide for intracellular protein delivery.

To facilitate the introduction of proteins, such as antibodies, into cells, a variety of delivery peptides have been engineered. These peptides are typically highly cationic and somewhat hydrophobic, enabling cytosolic protein delivery at the cost of causing cell damage by rupturing membranes. This balance between delivery effectiveness and cytotoxicity presents obstacles for their real-world use. To tackle this problem, we designed a new endosome-disruptive cytosolic delivery peptide, E3MPH16, inspired by mastoparan X (MP). E3MPH16 was engineered to incorporate three Glu (E3) and 16 His (H16) residues at the N- and C-termini of MP, respectively. The negative charges of E3 substantially mitigate the cell-surface damage induced by MP. The H16 segment is known to enhance cell-surface adsorption and endocytic uptake of the associated molecules. With these modifications, E3MPH16 was successfully trapped within endosomes. The acidification of endosomes is expected to protonate the side chains of E3 and H16, enabling E3MPH16 to rupture endosomal membranes. As a result, nearly 100% of cells achieved cytosolic delivery of a model biomacromolecule, Alexa Fluor 488-labeled dextran (10 kDa), via endosomal escape by co-incubation with E3MPH16. The delivery process also suggested the involvement of macropinocytosis and caveolae-mediated endocytosis. With the assistance of E3MPH16, Cre recombinase and anti-Ras-IgG delivered into HEK293 cells and HT1080 cells enabled gene recombination and inhibited cell proliferation, respectively. The potential for in vivo application of this intracellular delivery method was further validated by topically injecting the green fluorescent protein fused with a nuclear localization signal (NLS-GFP) along with E3MPH16 into Colon-26 tumor xenografts in mice.

A novel Menin-MLL1 inhibitor, DS-1594a, prevents the progression of acute leukemia with rearranged MLL1 or mutated NPM1.

BACKGROUND: Mixed lineage leukemia 1-rearranged (MLL1-r) acute leukemia patients respond poorly to currently available treatments and there is a need to develop more effective therapies directly disrupting the Menin‒MLL1 complex. Small-molecule-mediated inhibition of the protein‒protein interaction between Menin and MLL1 fusion proteins is a potential therapeutic strategy for patients with MLL1-r or mutated-nucleophosmin 1 (NPM1c) acute leukemia. In this study, we preclinically evaluated the new compound DS-1594a and its salts. METHODS: We evaluated the preclinical efficacy of DS-1594a as well as DS-1594a·HCl (the HCl salt of DS-1594a) and DS-1594a·succinate (the succinic acid salt of DS-1594a, DS-1594b) in vitro and in vivo using acute myeloid leukemia (AML)/acute lymphoblastic leukemia (ALL) models. RESULTS: Our results showed that MLL1-r or NPM1c human leukemic cell lines were selectively and highly sensitive to DS-1594a·HCl, with 50% growth inhibition values < 30 nM. Compared with cytrabine, the standard chemotherapy drug as AML therapy, both DS-1594a·HCl and DS-1594a·succinate mediated the eradication of potential leukemia-initiating cells by enhancing differentiation and reducing serial colony-forming potential in MLL1-r AML cells in vitro. The results were confirmed by flow cytometry, RNA sequencing, RT‒qPCR and chromatin immunoprecipitation sequencing analyses. DS-1594a·HCl and DS-1594a·succinate exhibited significant antitumor efficacy and survival benefit in MOLM-13 cell and patient-derived xenograft models of MLL1-r or NPM1c acute leukemia in vivo. CONCLUSION: We have generated a novel, potent, orally available small-molecule inhibitor of the Menin-MLL1 interaction, DS-1594a. Our results suggest that DS-1594a has medicinal properties distinct from those of cytarabine and that DS-1594a has the potential to be a new anticancer therapy and support oral dosing regimen for clinical studies (NCT04752163).

Functional lipidomics of vascular endothelial cells in response to laminar shear stress.

Laminar shear stress generated by blood flow stimulates endothelial cells and activates signal transduction, which plays an important role in vascular homeostasis. Several lines of evidence indicate that membrane and intracellular lipids are involved in the signal transduction of biomechanical stresses. In this study, we performed global profiling of cellular lipids from human pulmonary artery endothelial cells (HPAEC) exposed to laminar shear stress. A total of 761 species of lipids were successfully annotated, with 198 of these species significantly changed in response to shear stress for 24 hours. Ether-linked lipids containing an alkyl moiety with a medium chain length (C11-C14) were uniquely upregulated, and the administration of their biosynthetic precursor 1-O-dodecyl-rac-glycerol attenuated phorbol 12-myristate 13-acetate (PMA) induced vascular cell adhesion molecule-1 (VCAM-1) expression. Given the pro-inflammatory and atherogenic roles of VCAM-1, our findings suggest that the induction of a specific group of lipids (ie, ether-linked lipids with medium length alkyl side chain) may confer atheroprotective and anti-inflammatory roles to vascular endothelial cells under flow conditions.

Upregulated Kynurenine Pathway Enzymes in Aortic Atherosclerotic Aneurysm: Macrophage Kynureninase Downregulates Inflammation.

AIMS: Inflammation and hypertension contribute to the progression of atherosclerotic aneurysm in the aorta. Vascular cell metabolism is regarded to modulate atherogenesis, but the metabolic alterations that occur in atherosclerotic aneurysm remain unknown. The present study aimed to identify metabolic pathways and metabolites in aneurysmal walls and examine their roles in atherogenesis. METHODS: Gene expression using microarray and metabolite levels in the early atherosclerotic lesions and aneurysmal walls obtained from 42 patients undergoing aortic surgery were investigated (early lesion n=11, aneurysm n=35) and capillary electrophoresis-time-of-flight mass spectrometry (early lesion n=14, aneurysm n=38). Using immunohistochemistry, the protein expression and localization of the identified factors were examined (early lesion n=11, non-aneurysmal advanced lesion n=8, aneurysm n=11). The roles of the factors in atherogenesis were analyzed in macrophages derived from human peripheral blood mononuclear cells. RESULTS: Enrichment analysis using 35 significantly upregulated genes (log2 ratio, ＞3) revealed the alteration of the kynurenine pathway. Metabolite levels of tryptophan, kynurenine, and quinolinic acid and the kynurenine-to-tryptophan ratio were increased in the aneurysmal walls. Gene and protein expression of kynureninase and kynurenine 3-monooxygenase were upregulated and localized in macrophages in the aneurysmal walls. The silencing of kynureninase in the cultured macrophages enhanced the expression of interleukin-6 and indoleamine 2,3-dioxygenase 1. CONCLUSION: Our study suggests the upregulation of the kynurenine pathway in macrophages in aortic atherosclerotic aneurysm. Kynureninase may negatively regulate inflammation via the kynurenine pathway itself in macrophages.

Effects of a New Type of Shrinkage-Reducing Agent on Concrete Properties.

Shrinkage-reducing agents have been developed to mitigate shrinkage and to control cracks in concrete. This study aims to evaluate the impact of a newly developed shrinkage-reducing agent (N-SRA) on concrete properties and to compare its properties with a conventional shrinkage-reducing agent (C-SRA). The hydration rate, compressive strength, splitting tensile strength, shrinkage, occurrence of cracking, and freezing and thawing were investigated. N-SRA showed higher surface tension than C-SRA and reduced shrinkage to the same degree as C-SRA with half the dosage of C-SRA. The addition of N-SRA or C-SRA did not influence the early compressive strength but slightly reduced splitting tensile strength at seven days. Concrete with N-SRA showed higher compressive strength at 28 days than those of concrete with C-SRA or without SRA. Furthermore, concrete with N-SRA extended the period for the occurrence of shrinkage cracking under restrained conditions. It was found that N-SRA provided excellent freezing and thawing resistance because of the formation of good air voids, while C-SRA demonstrated inefficient behaviour in such an environment.

Adsorbed Conformations of PCE Superplasticizers in Cement Pore Solution Unraveled by Molecular Dynamics Simulations.

The conformations of polycarboxylate ether (PCE) type superplasticizer polymers adsorbed on the surface of MgO in cement pore solution are simulated by molecular dynamics (MD). Three types of PCEs commonly applied to concrete are simulated, namely a methacrylate type PCE (PCEM-P), an allyl ether type PCE (PCEA-P), and an isoprenyl ether type PCE (PCEI-P) with ethylene oxide (EO) unit numbers (P) of 25, 34 and 25, respectively. It is observed that the adsorbed layer thickness is inversely proportional to the experimentally measured adsorbed amount at the initial paste flow of 26 ± 0.5 cm. Simulation results indicate that the adsorbed layer thickness is sensitive to the initial polymer orientations against the model MgO surface. I.e., polymer molecules initially placed parallel/perpendicularly against the MgO surface gradually forms a train shaped or a loop and tail adsorption profile, respectively. As a result, the loop and tail shaped conformation gives a higher layer thickness.

Thioredoxin-1 maintains mechanistic target of rapamycin (mTOR) function during oxidative stress in cardiomyocytes.

Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds. As such, Trx1 helps protect the heart against stresses, such as ischemia and pressure overload. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, and survival. We have shown previously that mTOR activity is increased in response to myocardial ischemia-reperfusion injury. However, whether Trx1 interacts with mTOR to preserve heart function remains unknown. Using a substrate-trapping mutant of Trx1 (Trx1C35S), we show here that mTOR is a direct interacting partner of Trx1 in the heart. In response to H2O2 treatment in cardiomyocytes, mTOR exhibited a high molecular weight shift in non-reducing SDS-PAGE in a 2-mercaptoethanol-sensitive manner, suggesting that mTOR is oxidized and forms disulfide bonds with itself or other proteins. The mTOR oxidation was accompanied by reduced phosphorylation of endogenous substrates, such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1) in cardiomyocytes. Immune complex kinase assays disclosed that H2O2 treatment diminished mTOR kinase activity, indicating that mTOR is inhibited by oxidation. Of note, Trx1 overexpression attenuated both H2O2-mediated mTOR oxidation and inhibition, whereas Trx1 knockdown increased mTOR oxidation and inhibition. Moreover, Trx1 normalized H2O2-induced down-regulation of metabolic genes and stimulation of cell death, and an mTOR inhibitor abolished Trx1-mediated rescue of gene expression. H2O2-induced oxidation and inhibition of mTOR were attenuated when Cys-1483 of mTOR was mutated to phenylalanine. These results suggest that Trx1 protects cardiomyocytes against stress by reducing mTOR at Cys-1483, thereby preserving the activity of mTOR and inhibiting cell death.

Pathological and gene expression analysis of a polygenic diabetes model, NONcNZO10/LtJ mice.

The NONcNZO10/LtJ mouse is a polygenic model of type-2 diabetes (T2D) that shows moderate obesity and diabetes, and is regarded as a good model reflective of the conditions of human T2D. In this study, we analyzed pathological changes of pancreases with the progress of time by using histopathology and gene expression analysis, including microRNA. A number of gene expression changes associated with decreased insulin secretion (possibly regulated by miR-29a/b) were observed, and zinc homeostasis (Slc30a8, Mt1 and Mt2) or glucose metabolism (Slc2a2) was suggested as being the candidate mechanism of pancreas failure in NONcNZO10/LtJ mice. These results demonstrate NONcNZO10/LtJ mice have a complex pathogenic mechanism of diabetes, and moreover, this fundamental information of NONcNZO10/LtJ mice would offer the opportunity for research and development of a novel antidiabetic drug.

Lead substances selection using GHS approach for the classification of mixtures: Case study of painting in the work environment.

We developed a lead substances selection approach based on the concept of mixture classification of UN GHS for the purpose of efficient risk assessment of mixtures consisting of multiple components. Lead substances selection methods are being actively developed in Europe, but these methods are predicated on the regulations and information sources available within Europe and are therefore not readily applicable to countries outside Europe. In this study, the features of the GHS-based approach and the risk assessment results for outdoor painting work as a specific utilization example of the GHS-based approach were described. Comparison with the DPD + method and the CCA method proposed in Europe revealed that the GHS-based approach resulted in the selection of the safest lead substances. The GHS method, like the DPD + method, is a classification-based approach. We believe that a classification-based approach based on the GHS method can be an appropriate tool to efficiently implement risk assessment of mixtures for countries outside Europe. Some tools for business operators to conduct the management of chemicals using the GHS classification have been established in Japan. We plan to propose the GHS-based approach as a standardized assessment tool.

Transgenic overexpression of USP15 in the heart induces cardiac remodeling in mice.

We found a novel protein-protein interaction between ubiquitin-specific protease 15 (USP15) and skeletal muscle LIM protein 1 (SLIM1): USP15 and SLIM1 directly bound under cell-free conditions and co-immunoprecipitated from the lysates of the cells, where they were co-expressed; and USP15 deubiquitinated SLIM1, resulting in the increase of protein levels of SLIM1. Because SLIM1 is strongly implicated in the pathogenesis of myopathies and cardiomyopathies, we generated transgenic (TG) mice with cardiac-specific overexpression of human USP15. Heart weight to body weight ratios and mRNA levels of fetal gene markers in the heart were significantly higher in USP15-TG mice than in wild-type (WT) mice. Also, protein levels of endogenous murine SLIM1 in the heart were significantly higher in USP15-TG mice than in WT mice. Furthermore, the protein of alternatively spliced isoform of SLIM1 was only detected in the heart of USP15-TG mice, and mRNA levels of this isoform were higher as compared to WT mice. These results indicate that USP15 is involved in the regulation of hypertrophic responses in cardiac muscle through transcriptional and post-translational modulation of SLIM1.

Dual-targeting properties of the 3-aminopyrrolidyl quinolones, DC-159a and sitafloxacin, against DNA gyrase and topoisomerase IV: contribution to reducing in vitro emergence of quinolone-resistant Streptococcus pneumoniae.

OBJECTIVES: DC-159a (a novel quinolone) and sitafloxacin (DU-6859a) are structurally related quinolones, bearing a 3-aminopyrrolidyl substitution. We investigated the relationship between the target preferences of these 3-aminopyrrolidyl quinolones, in vitro potencies and emergence of quinolone-resistant mutants in Streptococcus pneumoniae, compared with other quinolones. METHODS: MICs, resistance frequencies and mutant prevention concentrations (MPCs) were determined using quinolone-susceptible strains and first-step parC mutant strains of S. pneumoniae. Target preferences were tested by the following two methods: antibacterial activities against gyrA or parC mutants and in vitro enzyme assays for the determination of 50% inhibition (IC(50)) values. RESULTS: DC-159a and sitafloxacin exhibited potent antibacterial activities, low frequencies of mutant selection, low MPCs and narrow mutant selection windows against both quinolone-susceptible strains and first-step parC mutants of S. pneumoniae, compared with gatifloxacin, moxifloxacin and other quinolones tested. DC-159a and sitafloxacin showed relatively low MIC ratios against single gyrA or parC mutants relative to the wild-type strain and low IC(50) ratios against DNA gyrase and topoisomerase IV. CONCLUSIONS: DC-159a and sitafloxacin demonstrated a more balanced dual-targeting activity than gatifloxacin, moxifloxacin and other quinolones tested. In addition, DC-159a and sitafloxacin have a lower propensity for selecting first- and second-step resistant mutants.

Quantitative risk assessment of noroviruses in drinking water based on qualitative data in Japan.

Noroviruses are one of the major causes of viral gastroenteritis in Japan. A quantitative risk assessment was conducted to evaluate the health risk caused by this virus in drinking water. A Monte Carlo analysis was used to calculate both the probability of infection and the disease burden using disability-adjusted life years (DALYs). The concentration of noroviruses in tap water was estimated based on qualitative data and a most probable number (MPN) method with an assumed Poisson lognormal distribution. This numerical method was evaluated using two sets of available count data of Cryptosporidium: that collected from a river and that found in tap water in Japan. The dose-response relationships for noroviruses were estimated using assumed ID50 (10 or 100). The annual risk was higher than the US-EPA acceptable level (10(-4) [infection/ person-year]) but around the WHO level (10(-6) [DALYs/ person-year]). As suggested by others, since microbial concentrations are generally lognormally distributed, the arithmetic mean was directly related to the annual risk, suggesting that the arithmetic mean is more useful in representing the degree of microbial contamination than the geometric mean.

Genotyping of single Cryptosporidium oocysts in sewage by semi-nested PCR and direct sequencing.

This study describes an approach for genotyping individual Cryptosporidium oocysts obtained from sewage. We isolated single immunofluorescent assay (IFA)-stained Cryptosporidium oocysts from sewage concentrate using glass capillary pipettes and inverted epifluorescence microscopy. Each isolated Cryptosporidium oocyst was analyzed by semi-nested PCR for the 18S rRNA gene and direct sequencing of the PCR products. A total of 74 of 107 oocysts isolated from sewage were genotyped successfully. Of the 74 genotyped isolates, 51% (38 oocysts) were identified as C. parvum genotype 1, 4% (3 oocysts) of C. parvum VF383 human isolates, 20% (15 oocysts) of C. parvum genotype 2, 14% (10 oocysts) of C. meleagridis, 7% (5 oocysts) of C. sp. Pig 1, 3% (2 oocysts) of C. sp PG1-26 pig isolates and 1% (1 oocyst) of C. parvum CPM1 isolated from mouse. The results of this study demonstrate that 18S rRNA-based semi-nested PCR and direct sequencing can be used to characterize individual Cryptosporidium oocysts and also to reveal the distribution of Cryptosporidium genotypes in environmental waters.

Biomolecular mirror-image recognition: reciprocal chiral-specific DNA binding of synthetic enantiomers of zinc finger domain from GAGA factor.

To experimentally demonstrate the mirror-image recognition in protein and DNA interaction, we have designed a small DNA-binding peptide based on the zinc-finger domain of GAGA transcription factor. Circular dichroism data suggest that the conformations of peptide enantiomers as well as the DNA enantiomers have a mirror-image relationship. The gel mobility shift assay showed that the synthetic enantiomers of the peptide showed reciprocal chiral-specific interactions with the DNA; the natural L-peptide binds specifically with the natural D-DNA substrate, and the unnatural D-peptide binds specifically with the unnatural L-DNA substrate. The present data imply that the folding of the L- and D-enantiomers of the peptide as well as the DNA substrates are exact mirror images of each other in 3-D structure and biological activity, and generalize the chiral-specific nature of biomolecular interaction.

Effects of linking 15-zinc finger domains on DNA binding specificity and multiple DNA binding modes.

To assess the possibility of multi-connection of zinc finger domains for understanding of DNA binding mechanisms and gene regulation, the longest artificial zinc finger protein, Sp1ZF15, has been constructed. This zinc finger consists of 5 units of Sp1 zinc finger peptide connected by canonical short linker sequences (TGEKP). Recognition of the 50 base pairs of DNA and potential binding to shorter targets by Sp1ZF15 were determined. Sequence alterations of the GCG triplet to ATA at a target site clearly showed that Sp1ZF15 changes its DNA binding mode depending on the target sequences. Of special interest is the fact that Sp1ZF15 controls the number of finger domains active in DNA binding corresponding to the length and sequence of the target DNA. These results suggest that artificial transcription factors based upon these multi-zinc finger proteins have great potential for the regulation of a vast number of cellular processes.

Efficacy of UV irradiation in inactivating Cryptosporidium parvum oocysts.

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log(10) reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm(2) at 20 degrees C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm(2) for a 2-log(10) reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm(2). Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10 degrees C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log(10) reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.

Prevalence of Cryptosporidium oocysts and giardia cysts in the drinking water supply in Japan.

A one-year monitoring of Cryptosporidium oocysts and Giardia cysts was conducted at a water purification plant. A total of 13 samples of 50 L river source water and 26 samples of 2,000 L-filtered water, treated by coagulation flocculation, sedimentation and rapid filtration, were tested. Prior to conducting a survey of a water purification plant, we developed a method for concentrating Cryptosporidium oocysts from a large volume of raw or filtered water using a hollow fiber ultrafiltration (UF) membrane, and this procedure was adapted to survey a water purification plant. Cryptosporidium oocysts were detected in all of the 13 raw water samples. The geometric mean concentration was 40 oocysts 100 L. Giardia cysts were detected in 12 of 13 raw water samples (92%) and the geometric mean concentration was 17 cysts/100 L. Probability distributions of both Cryptosporidium oocyst and Giardia cyst concentration in raw water were nearly lognormal. In filtered water samples, Cryptosporidium oocysts were detected in 9 of the 26 samples (35%) with the geometric mean concentration of 1.2 oocysts /1,000 L and Giardia cysts in 3 samples (12%) with 0.8 cysts/1,000 L. The estimated log10 removal efficiency of Cryptosporidium oocysts and Giardia cysts by rapid-sand filtration was 2.47 and 2.53, respectively. Empty particles were removed at a higher log10 than intact oocysts and cysts. The efficiency of particle removal in the rapid sand filtration process tends to be reduced under cold-water conditions. Close management is necessary in the winter when the water temperature is low.