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Three RpoD-family sigma factors, RpoD, RpoS, and RpoH, play critical roles in transcriptional regulation in Salmonella enterica serovar Typhimurium under heat shock conditions. However, the genome-wide regulatory mechanisms of these sigma factors in response to heat stress have remained elusive. In this study, we comprehensively identified 2,319, 2,226, and 213 genome-wide binding sites for RpoD, RpoS, and RpoH, respectively, under sublethal heat shock conditions (42°C). Machine learning-based transcriptome analysis was employed to infer the relative activity of iModulons, providing valuable insights into the transcriptional impact of heat shock. Integrative data analysis enabled the reconstruction of the transcriptional regulatory network of sigma factors, revealing how they modulate gene expression to adapt to heat stress, including responses to anaerobic and oxidative stresses. Notably, we observed a significant expansion of the RpoS sigmulon from 97 to 301 genes in response to heat shock, underscoring the crucial role of RpoS in regulating various metabolic processes. Moreover, we uncovered a competition mechanism between RpoD and RpoS within RpoS sigmulons, where RpoS significantly increases its binding within promoter regions shared with RpoD under heat shock conditions. These findings illuminate how three RpoD-family sigma factors coordinate multiple cellular processes to orchestrate the overall response of S. Typhimurium to heat stress.

ABSTRACTAntimicrobial resistance (AMR) in pathogenic bacteria poses a significant threat to public health, yet there is still a need for development in the tools to deeply understand AMR genes based on genetic or structural information. In this study, we present an interactive web database named Blanket Overarching Antimicrobial-Resistance gene Database with Structural information (BOARDS, sbml.unist.ac.kr), a database that comprehensively includes 3,943 reported AMR gene information for 1,997 extended spectrum beta-lactamase (ESBL) and 1,946 other genes as well as a total of 27,395 predicted protein structures. These structures, which include both wild-type AMR genes and their mutants, were derived from 80,094 publicly available whole-genome sequences. In addition, we developed the rapid analysis and detection tool of antimicrobial-resistance (RADAR), a one-stop analysis pipeline to detect AMR genes across whole-genome sequencing (WGSs). By integrating BOARDS and RADAR, the AMR prevalence landscape for eight multi-drug resistant pathogens was reconstructed, leading to unexpected findings such as the pre-existence of the MCR genes before their official reports. Enzymatic structure prediction-based analysis revealed that the occurrence of mutations found in some ESBL genes was found to be closely related to the binding affinities with their antibiotic substrates. Overall, BOARDS can play a significant role in performing in-depth analysis on AMR.IMPORTANCEWhile the increasing antibiotic resistance (AMR) in pathogen has been a burden on public health, effective tools for deep understanding of AMR based on genetic or structural information remain limited. In this study, a blanket overarching antimicrobial-resistance gene database with structure information (BOARDS)—a web-based database that comprehensively collected AMR gene data with predictive protein structural information was constructed. Additionally, we report the development of a RADAR pipeline that can analyze whole-genome sequences as well. BOARDS, which includes sequence and structural information, has shown the historical landscape and prevalence of the AMR genes and can provide insight into single-nucleotide polymorphism effects on antibiotic degrading enzymes within protein structures.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common foodborne pathogen which is frequently used as the reference strain for Salmonella. Investigating the sigma factor network and protomers is crucial to understand the genomic and transcriptomic properties of the bacterium. Its promoters were identified using various methods such as dRNA-seq, ChIP-chip, or ChIP-Seq. However, validation using ChIP-exo, which exhibits higher-resolution performance compared to conventional ChIP, has not been conducted to date. In this study, using the representative strain S. Typhimurium LT2 (LT2), the ChIP-exo experiment was conducted to accurately determine the binding sites of catalytic RNA polymerase subunit RpoB and major sigma factors (RpoD, RpoN, RpoS, and RpoE) during exponential phase. Integrated with the results of RNA-Seq, promoters and sigmulons for the sigma factors and their association with RpoB have been discovered. Notably, the overlapping regions among binding sites of each alternative sigma factor were found. Furthermore, comparative analysis with Escherichia coli str. K-12 substr. MG1655 (MG1655) revealed conserved binding sites of RpoD and RpoN across different species. In the case of small RNAs (sRNAs), 50 sRNAs observed their expression during the exponential growth of LT2. Collectively, the integration of ChIP-exo and RNA-Seq enables genome-scale promoter mapping with high resolution and facilitates the characterization of binding events of alternative sigma factors, enabling a comprehensive understanding of the bacterial sigma factor network and condition-specific active promoters.

Abstract Transcranial focused ultrasound stimulation is a promising therapeutic modality for human brain disorders because of its noninvasiveness, long penetration depth, and versatile spatial control capability through beamforming and beam steering. However, the skull presents a major hurdle for successful applications of ultrasound stimulation. Specifically, skull-induced focal aberration limits the capability for accurate and versatile targeting of brain subregions. In addition, there lacks a fully functional preclinical neuromodulation system suitable to conduct behavioral studies. Here, we report a miniature ultrasound system for neuromodulation applications that is capable of highly accurate multiregion targeting based on acoustic holography. Our work includes the design and implementation of an acoustic lens for targeting brain regions with compensation for skull aberration through time-reversal recording and a phase conjugation mirror. Moreover, we utilize MEMS and 3D-printing technology to implement a 0.75-g lightweight neuromodulation system and present in vivo characterization of the packaged system in freely moving mice. This preclinical system is capable of accurately targeting the desired individual or multitude of brain regions, which will enable versatile and explorative behavior studies using ultrasound neuromodulation to facilitate widespread clinical adoption.

Abstract Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic debilitating disease in ruminants. To control this disease, it is crucial to understand immune evasion and the mechanism of persistence by analyzing the early phase interplays of the intracellular pathogens and their hosts. In the present study, host–pathogen interactions at the transcriptomic level were investigated in an in vitro macrophage infection model. When differentiated human THP-1 cells were infected with MAP, the expression of various genes associated with stress responses and metabolism was altered in both host and MAP at 3 h post-infection. MAP upregulates stress-responsive global gene regulators, such as two-component systems and sigma factors, in response to oxidative and cell wall stress. Downstream genes involved in type VII secretion systems, cell wall synthesis (polyketide biosynthesis proteins), and iron uptake were changed in response to the intracellular environment of macrophages. On the host side, upregulation of inflammatory cytokine genes was observed along with pattern recognition receptor genes. Notably, alterations in gene sets involved in arginine metabolism were observed in both the host and MAP, along with significant downregulation of NOS2 expression. These observations suggest that the utilization of metabolites such as arginine by intracellular MAP might affect host NO production. Our dual RNA-seq data can provide novel insights by capturing the global transcriptome with higher resolution, especially in MAP, thus enabling a more systematic understanding of host–pathogen interactions.

The energy-intensive steel industry, which consumes substantial amounts of electricity, meets its power demands through external electricity purchases and self-generation through the operation of its own generators. This study aimed to optimize boiler combustion efficiency and increase power generation output by deriving optimal operational values for O2 and CO within the boiler flue gas using machine learning (ML) with the aim of achieving maximum boiler efficiency. This study focuses on the power-generation boilers at steel mill P in Korea. First, 361 types of operation data from power generation equipment were collected and preprocessed. Subsequently, a partial least squares regression (PLSR) algorithm was used to develop a prediction model for O2 and CO values, known as the Boiler Flue Gas Prediction Model (BFG-PM). The prediction accuracy for O2 was notably high (83.2%), whereas that for CO was lower (53.4%). Nonetheless, the model’s reliability was high because more than 90% of the predicted values were within a 10% error range. Finally, the correlation of the BFG-PM model was applied to the performance test code (PTC) 4.0 for the boiler efficiency calculations formula, deriving the optimal O2 and CO control points. Through a simulation, it was verified that the boiler efficiency was improved by controlling the combustion air. In addition, an average increase in boiler efficiency of 0.29% was confirmed by applying it directly to the generator operating on-site. The results of this study are expected to contribute to annual cost savings, with a reduction of USD 217,000 in electricity purchasing costs and USD 19,700 in greenhouse gas emissions trading expenses.

ABSTRACT Although Mycobacterium avium subsp. paratuberculosis (MAP) has threatened public health and the livestock industry, the current diagnostic tools (e.g., fecal PCR and enzyme-linked immunosorbent assay [ELISA]) for MAP infection have some limitations, such as inconsistent results due to intermittent bacterial shedding or low sensitivity during the early stage of infection. Therefore, this study aimed to develop a novel biomarker focusing on elucidating the gut microbial signature of MAP-positive ruminants, since the clinical signs of MAP infection are closely related to dysbiosis. 16S rRNA-based gut microbial community analysis revealed both a decrease in microbial diversity and the emergence of several distinct taxa following MAP infection. To determine the discriminant taxa diagnostic of MAP infection, machine learning-based feature selection and predictive model construction were applied to taxon abundance data or their transformed derivatives. The selected taxa, such as Clostridioides (formerly Clostridium) difficile, were used to build models using a support vector machine, linear support vector classification, k-nearest neighbor, and random forest with 10-fold cross-validation. The receiver operating characteristic-area under the curve (ROC-AUC) analysis of the models revealed their high accuracy, up to approximately 96%. Collectively, taxonomic signatures of cattle gut microbiotas according to MAP infection status could be identified by feature selection tools and applied to establish a predictive model for the infection state. IMPORTANCE Due to the limitations, such as intermittent bacterial shedding or poor sensitivity, of the current diagnostic tools for Johne’s disease, novel biomarkers are urgently needed to aid control of the disease. Here, we explored the fecal microbiota of Johne’s disease-affected cattle and tried to discover distinct microbial characteristics which have the potential to be novel noninvasive biomarkers. Through 16S rRNA sequencing and machine learning approaches, a dozen taxa were selected as taxonomic signatures to discriminate the disease state. In addition, when constructing predictive models using relative abundance data of the corresponding taxa, the models showed high accuracy for classification, even including animals with subclinical infection. Thus, our study suggested novel noninvasive microbiological biomarkers that are robustly expressed regardless of subclinical infection and the applicability of machine learning for diagnosis of Johne’s disease.

Concerns over Klebsiella pneumoniae resistance to the last-line antibiotic treatment have prompted a reconsideration of bacteriophage therapy in public health. Biotechnological application of phages and their gene products as an alternative to antibiotics necessitates the understanding of their genomic context. This study sequenced, annotated, characterized, and compared two Klebsiella phages, KP1 and KP12. Physiological validations identified KP1 and KP12 as members of Myoviridae family. Both phages showed that their activities were stable in a wide range of pH and temperature. They exhibit a host specificity toward K. pneumoniae with a broad intraspecies host range. General features of genome size, coding density, percentage GC content, and phylogenetic analyses revealed that these bacteriophages are distantly related. Phage lytic proteins (endolysin, anti-/holin, spanin) identified by the local alignment against different databases, were subjected to further bioinformatic analyses including three-dimensional (3D) structure prediction by AlphaFold. AlphaFold models of phage lysis proteins were consistent with the published X-ray crystal structures, suggesting the presence of T4-like and P1/P2-like bacteriophage lysis proteins in KP1 and KP12, respectively. By providing the primary sequence information, this study contributes novel bacteriophages for research and development pipelines of phage therapy that ultimately, cater to the unmet clinical and industrial needs against K. pneumoniae pathogens.

Abstract Transcranial focused ultrasound stimulation (tFUS) is an effective noninvasive treatment modality for brain disorders with high clinical potential. However, the therapeutic effects of ultrasound neuromodulation are not widely explored due to limitations in preclinical systems. The current preclinical studies are head‐fixed, anesthesia‐dependent, and acute, limiting clinical translatability. Here, this work reports a general‐purpose ultrasound neuromodulation system for chronic, closed‐loop preclinical studies in freely behaving rodents. This work uses microelectromechanical systems (MEMS) technology to design and fabricate a small and lightweight transducer capable of artifact‐free stimulation and simultaneous neural recording. Using the general‐purpose system, it can be observed that state‐dependent ultrasound neuromodulation of the prefrontal cortex increases rapid eye movement (REM) sleep and protects spatial working memory to REM sleep deprivation. The system will allow explorative studies in brain disease therapeutics and neuromodulation using ultrasound stimulation for widespread clinical adoption.

Abstract To report the efficacy and safety of large diameter scleral lenses and determine their suitability in Asian subjects with intractable ocular surface diseases. This prospective study enrolled intractable ocular surface diseases subjects with uncorrected visual acuity > counting finger but ≥ 0.3 logMAR and best-corrected visual acuity (BCVA) ≥ 0.3 logMAR, to fit large diameter scleral lenses for 12 weeks. 21 eyes (13 subjects) consisting ten eyes (47.6%) with persistent epithelial defects, 6 (28.6%) with graft-versus-host disease, 4 (19.0%) with Stevens–Johnson syndrome and one (4.8%) with severe dry eye were ultimately enrolled. Primary outcome measures were the visual acuity, corneal and conjunctival fluorescein staining, Ocular Surface Disease Index (OSDI), and National Eye Institute 25-Item Visual Function Questionnaire (NEI-VFQ-25). At week 12 with large diameter scleral lenses, BCVA improved from 0.77 logMAR to 0.27 logMAR (P

Although polyunsaturated fatty acids (PUFAs) have gained attention because of their physiological effects on inflammation and stress, their derivatives, termed lipid mediators, have been rarely examined, especially in laying hens. In this study, we aimed to investigate PUFA and lipid mediator profiles of laying hens after feeding 0, 0.9, 1.8, or 3.6% (w/w) dietary flaxseed (Lintex170) for 4 weeks. We also monitored the indices of inflammation (serum proinflammatory cytokines) and stress (serum corticosterone, ratio of heterophils to lymphocytes). In this study, flaxseed reduced the omega-6 to omega-3 FA ratio and increased several omega-3 FA-derived lipid mediators. Furthermore, levels of the proinflammatory cytokine tumour necrosis factor alpha and two stress indices (corticosterone, heterophil to lymphocyte ratio) were decreased when fed the 3.6% flaxseed diet. Overall, laying performance indices were also significantly improved by flaxseed treatment. These findings suggest that flaxseed may alleviate the stress state of laying hens by enriching omega-3-derived lipid mediators, which could improve laying performance.

A new formulation, nanoprebiotics [e.g., phthalyl pullulan nanoparticles (PPNs)], was demonstrated to enhance the antimicrobial activity of probiotics [e.g., Lactobacillus plantarum (LP)] in vitro through intracellular stimulation better than that by backbone prebiotics, which are commonly used. In this study, we aimed to investigate whether this combination would exert distinct effects as synbiotics in vivo. Synbiotics combinations of LP, pullulan, and PPNs were used as experimental treatments in a dysbiosis-induced murine model, and their restorative effect was assessed using pathogen Escherichia coli K99 challenge. Our results showed that the E. coli infection was suppressed markedly in the experimental group fed with synbiotics containing PPNs. In addition, the decrease in serum endotoxin level after synbiotics treatment suggested the reinforcement of the gut barrier. Comparison of treatment groups, including a normal control group, showed that synbiotics containing PPNs increased microbial diversity, which is a representative parameter of healthy status. Furthermore, distinct from probiotics treatment alone, synbiotics showed additive effects of enrichment of several well-known beneficial bacteria such as Lactobacillus, Bifidobacterium, and other butyrate-producing bacteria including Faecalibacterium. Collectively, our results indicate that synbiotics containing PPNs are effective at restoring gut dysbiosis, suppressing pathogenic infection, and increasing microbial diversity, suggesting that synbiotics with nanoprebiotics have the potential to be a novel strategy for ameliorating gut dysbiosis and infectious diseases.

An acoustic matching layer is an essential component of an ultrasound transducer to achieve maximum ultrasound transmission efficiency. Here, we develop a flexible printed circuit board (FPCB) with a composite structure consisting of multiple polyimide and copper layers and demonstrate it as a novel acoustic matching layer. With a flexible substrate and robust ACF bonding, the FPCB not only serves as an acoustic matching layer between piezoelectric elements and the surrounding medium but also as a ground for the electrical connection between the transducer array elements and the folded substrate. A 1D linear ultrasound transducer array with the FPCB matching layer exhibits larger output pressure, wider -3dB bandwidth, and higher ultrasound beam intensity compared to that of an ultrasound transducer array with the alumina/epoxy matching layer, which is one of the most commonly applied composite matching layers. The enhanced transmission performance verifies that the proposed FPCB is an excellent matching layer for 1D linear ultrasound transducer arrays.

Abstract The association of visual impairment and depression has been investigated in several studies based on a cross-sectional design, which cannot delineate temporal relationships. In the present study, we evaluated the influence of visual impairment on depression in all age groups using a longitudinal database of a national sample cohort from 2002 to 2013 provided by the Korean National Health Insurance Service. Of a total of 1,025,340 subjects, 5,846 participants who were registered as visually impaired persons without a previous diagnosis of depression were enrolled at a 1:4 ratio with 23,384 control participants matched for age, sex, income, and region of residence. The crude and adjusted (age, sex, income, region of residence, hypertension, diabetes, and dyslipidemia) hazard ratios (HRs) for the development of depression between the visually impaired and control groups were analyzed using a Cox proportional hazards model. Visual impairment increased the risk of depression after adjusting for age, sex, income, region of residence, hypertension, diabetes, and dyslipidemia (adjusted HR = 1.19, P = 0.002). The risk of depression increased significantly in both the non-blindness visual impairment (adjusted HR = 1.15, P = 0.036) and blindness subgroups (adjusted HR = 1.31, P = 0.016), with a higher HR in the blindness subgroup.

Synbiotics, which are the combination of probiotics and prebiotics, have recently attracted attention because of their synergistic net health benefits. Probiotics have been used as alternatives to antibiotics. Among the probiotics, Lactobacillus plantarum (LP) has shown strong antimicrobial activity against Escherichia coli K99, a major livestock pathogen. In this study, we aimed to investigate the antimicrobial activity of phthalyl pullulan nanoparticle (PPN)-treated LP. Interestingly, when PPNs were added to LP, the PPNs were internalized into the LP through an energy-dependent and galactose transporter-dependent mechanism. Additionally, more plantaricin, a natural antibacterial peptide, was secreted from PPN-treated LP than from untreated or pullulan-treated LP. Furthermore, antimicrobial activity against Gram-negative Escherichia coli K99 and Gram-positive Listeria monocytogenes by PPN-treated LP was higher than those of untreated or pullulan-treated LP. It is thought that the enhanced antimicrobial properties of the PPN-treated LP are due to intracellular stimulation. Overall, this research provides a new method of producing plantaricin in LP through intracellular stimulation by internalized PPNs.

Purpose: C-reactive protein (CRP) has been identified to be associated with cancer development and progression. However, the role of CRP in tumor recurrence is less well defined in biliary tract cancer. Therefore, the aim of this study was to determine the clinical importance of CRP in terms of recurrence of biliary tract cancer after surgical resection. Methods: Sevent-six patients who underwent curative resection for biliary tract cancer from April 2006 to July 2014 were reviewed. Demographics and tumor characteristics were evaluated retrospectively by review of a clinical database and review of pathologic reports. Results: The study group was comprised of 48 men and 28 women, with a mean age of 62.2±10.8 years. Clinical diagnoses of these patients included intrahepatic cholangiocarcinoma (n=6), hilar cholangiocarcinoma (n=13), extrahepatic cholangiocarcinoma (n=21), gall bladder cancer (n=11), and ampullary cancer (n=25). Median follow-up was 35 months. Recurrence-free survival at 1-, 2-, and 5-year was 89%, 76%, and 53%, respectively. On univariate analysis, high CRP level (>3.0 mg/dL) was a significant risk factors for recurrence. Multivariate analysis showed high CRP, poor tumor differentiation, vascular invasion were independent risk factor for tumor recurrence. Conclusion: Preoperative CRP level is significantly associated with recurrence in patients with biliary tract cancer, suggesting the possibility of using CRP as an independent prognostic indicator for biliary cancer.

Purpose. To compare the eyelid pressure between patients with functional nasolacrimal duct obstruction (FNLDO) and normal controls using blepharo-tensiometer, and to evaluate the relationship between eyelid pressure and the outcomes of silicone intubation (SI) in patients with FNLDO. Study design. Prospective case-control study. Methods. We enrolled 36 eyes of 36 patients with suspected FNLDO who underwent SI and 36 healthy eyes of age-matched controls. One eye of each patient with FNLDO was randomly selected for analysis. The eyelid pressure was estimated using a blepharo-tensiometer and compared between the control and FNLDO groups. The relationship between eyelid pressure and clinical variables was analyzed. The outcomes of SI were assessed at 6 months after surgery using subjective and objective criteria. Results. The eyelid pressure was significantly lower in the FNLDO group than in the control group P=0.008. In the control group, the eyelid pressure was correlated with age P

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, the feeding environment, including the composition of animal intestinal microbiota, has changed rapidly. We hypothesized that the microbial genomes have also been affected by this legal prohibition, and investigated an important member of the swine gut microbiota, Lactobacillus salivarius, with a pan-genomic approach. Here, we isolated 21 L. salivarius strains composed of 6 strains isolated before the AGP prohibition (SBPs) and 15 strains isolated after the AGP prohibition (SAPs) at an interval of a decade, and the draft genomes were generated de novo. Several genomic differences between SBPs and SAPs were identified, although the number and function of antibiotic resistance genes were not different. SBPs showed larger genome size and a higher number of orthologs, as well as lower genetic diversity, than SAPs. SBPs had genes associated with the utilization of L-rhamnose and D-tagatose for energy production. Because these sugars are also used in exopolysaccharide (EPS) synthesis, we tried to identify differences in biofilm formation-associated genes. The genes for the production of EPSs and extracellular proteins were different in terms of amino acid sequences. Indeed, SAPs formed dense biofilm and survived better than SBPs in the swine intestinal environment. These results suggest that SAPs have evolved and adapted to protect themselves from new selection pressure of the swine intestinal microenvironment by forming dense biofilms, adopting a distinct antibiotic resistance strategy. This finding is particularly important to understand the evolutionary changes in host-microbe interaction and provide detailed insight for the development of effective probiotics for livestock.

TIMP-2 protein has been intensively studied as a promising anticancer candidate agent, but the in vivo mechanism underlying its anticancer effect has not been clearly elucidated by previous works. In this study, we investigated the mechanism underlying the anti-tumor effects of a TIMP-2 fusion protein conjugated with human serum albumin (HSA/TIMP-2). Systemic administration of HSA/TIMP-2 effectively inhibited tumor growth at a minimum effective dose of 60 mg/kg. The suppressive effect of HSA/TIMP-2 was accompanied by a marked reduction of in vivo vascularization. The anti-angiogenic activity of HSA/TIMP-2 was directly confirmed by CAM assays. In HSA/TIMP-2-treated tumor tissues, MMP-2 expression was profoundly decreased without a change in MT1-MMP expression of PECAM-1-positive cells. MMP-2 mRNA was also decreased by HSA/TIMP-2 treatment of human umbilical vein endothelial cells. Zymographic analysis showed that HSA/TIMP-2 substantially decreased extracellular pro-MMP-2 activity (94-99% reduction) and moderately decreased active MMP-2 activity (10-24% reduction), suggesting MT1-MMP-independent MMP-2 modulation. Furthermore, HSA/TIMP-2 had no effect on in vitro active MMP-2 activity and in vivo MMP-2 activity. These studies show that HSA/TIMP-2 potentiates anti-angiogenic activity by modulating MMP-2 expression, but not MMP-2 activity, to subsequently suppress tumor growth, suggesting an important role for MMP-2 expression rather than MMP-2 activity in anti-angiogenesis.

Purpose TIMP-2 has been studied as an attractive cancer therapeutic candidate, and a TIMP-2 fusion protein (HSA/TIMP-2) displayed effective anticancer activity, despite a lack of information about its pharmacokinetics (PK) and biodistribution. The purpose of this work was to assess the PK and biodistribution of HSA/TIMP-2 as well as to quantify accumulated HSA/TIMP-2 in tumors. Methods Cy5.5 near-infrared (NIR) fluorescence was conjugated to the HSA/TIMP-2 protein (Cy5.5–HSA/TIMP-2) for monitoring spatio-temporal changes in vivo. For PK and biodistribution analysis, 0.2 μg/g body weight of Cy5.5–HSA/TIMP-2 was injected into MAT-LyLu prostate tumor xenografts, which were then imaged using an IVIS-200 optical imaging system. To quantify the accumulated HSA/TIMP-2 in tumors, we introduced a standard curve with depth-corrected fluorescence measurement. Results In the vascular tube formation assay with human umbilical vein endothelial cells (HUVECs), Cy5.5–HSA/TIMP-2 showed an antiangiogenic effect. In prostate cancer xenografts, Cy5.5–HSA/TIMP-2 exhibited a prolongation of blood half-life to 19.6 h and relatively preferential distribution to the tumor. The amount of tumor-accumulated Cy5.5–HSA/TIMP-2 was calculated to be 4.5 ± 0.5 ng/g body weight at 2 days, representing 2.25 ± 0.25% of the initial dose. Conclusions We evaluated the pharmacokinetic profile and biodistribution of HSA/TIMP-2 with favorable results, providing new information for more effective approaches to cancer therapeutics using HSA/TIMP-2. Additionally, real-time in vivo fluorescence imaging analysis using a depth-corrected standard curve may serve as a platform to quantify biodistributed drug in anticancer therapeutic studies. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.